Bioactive peptides

ABSTRACT

The present invention provides a modified lactoferrin peptide which is cytotoxic, 7 to 25 amino acids in length, with three or more cationic residues and which has one or more extra bulky and lipophilic amino acids as compared to the native lactoferrin sequence, as well as esters, amides, salts and cyclic derivatives thereof as well as methods of preparing such peptides, pharmaceutical compositions containing such peptides and use of the peptides as medicaments, particularly as antibacterials or anti-tumoural agents.

[0001] The present invention relates to bioactive peptides, moreparticularly to peptides which have been modified to enhance theircytotoxic activity.

[0002] A wide variety of organisms use peptides as part of their hostdefense mechanism, in vertebrates this supplements the highly specificcell-mediated immune system [Mor, a., Hani, K. and Nicolas, P. (1994) J.Biol. Chem. 269, 31635-31641. Boman, H. G. (1996) Scand. J. Immunol. 43,475-482]. Antimicrobial peptides have been isolated from species asdiverse as bacteria and mammals [Lehrer, R. I., Lichtenstein, A. K. andGanz, T. (1993) Ann. Rev. Immunol. 11, 105-128] Generally, theseantibiotic peptides have a net positive charge and a propensity to formamphiphilic α-helix or β-sheet structures upon interaction with theouter phospholipid bilayer in bacterial cell membranes [Besalle, R.,Gorea, A., Shalit, J., Metger, J. W., Dass, C. Desiderio, D. M. andFridkin, M. (1993) J. Med. Chem. 36 1203-1209]. In most cases thedetailed molecular mechanisms of the antibiotic action are unknown,although some peptides categorised as class L (lytic) peptides arebelieved to interact with bacterial cell membranes, probably formingion-channels or pores [Ludtke, S. J., He, K., Heller, W. T., Harroun, T.A., Yang, L. and Huang, H. W. (1996) Biochemistry 35 13723-13728]leading to permeability changes and consequent cell lysis.

[0003] Magainins are antibacterial peptides from the skin of the frogXenopus laeris and are classified as class L antibiotics because theyspecifically lyse bacteria; other peptides such as mastroparans, a beevenom, lack this specificity as they lyse eukaryotic as well asprokaryotic cells and are called Class L Venoms [Tytler, E. M.,Anantharamaiah, G. M., Walker, D. E., Mishra, V. K., Palgunachari, M. N.and Segrest, J. P. (1995) Biochemistry 34 4393-4401]. Anti-bioticresistance exhibited by certain infectious microorganisms is anincreasing problems and there is always a need for new antibiotics.Anti-bacterial peptides such as the class L peptides are known and moreare being discovered, with the aim of finding a peptide which is highlycytotoxic and preferably specific for prokaryotic cells. There aredifferences in the structure and composition of lipid bi-layers betweeneukaryotes and prokaryotes and amongst prokaryotes themselves which meanthat different peptides will have widely differing specificities.

[0004] As well as magainins and mastroparans, host defense peptides havebeen isolated from moths and flies (cecropins) and from Horseshoe crab.The direct action of these host defense peptides to repel predators, forexample as venoms, is clear. The search for peptides which exhibitantibiotic effects has lead to the identification of otherproteins/peptides which would not be expected to have cytotoxicproperties. One of these is lactoferrin, an iron transporter which alsoshows a weak antibacterial effect.

[0005] As well as searching for new antimicrobial peptides, morerecently it has been sought to enhance the activity of proteins orpeptides with known antimicrobial properties. This has been done in thecase of bovine lactoferrin by digesting the native protein with gastricpepsin to produce a peptide, lactoferricin B (LFB), which is much moreactive than the native bovine lactoferrin. LFB is a 25 residue peptidewhich corresponds to residues 17-41 of bovine lactoferrin. [Bellamy etal. (1992) Biochem. Biophys. Acta. 1121 pp 130 et seq.].Structure-activity studies have been carried out on magainins and it hasbeen shown, for example, that enhancement of helicity and of thecationic charge leads to higher antibacterial activity [Chen, Y. H.,Brown, J. H., Morell, J. L. and Huang, C. M. (1988) FEBS Letters 236,462-466]. However, such sequence modifications often result in higherhemolytic activity. It is thus an object of the present invention toprepare peptides and/or peptide derivatives which have significantantibacterial activity but preferably have low toxicity, i.e. littleeffect on normal eukaryotic cells, e.g. low hemolytic activity. Whilered blood cells may not be typical eukaryotic cells, they provide aconvenient way of assaying for toxicity and in any event are a type ofcell which should not be lysed to a significant extent by therapeuticbioactive peptides.

[0006] It has been found that by increasing the bulk or lipophilicnature of a peptide derived from lactoferrin, its bioactivity can beincreased, in particular its cytotoxicity. Preferably, the bulk andlipophilicity of one or more amino acid residues is increased.

[0007] Thus, according to the present invention is provided a modifiedlactoferrin peptide which is cytotoxic, 7 to 25 amino acids in length,with three or more cationic residues which is optionally capable offorming an amphiphatic a-helix and which has one or more extra bulky andlipophilic amino acids as compared to the native lactoferrin sequence,as well as esters, amides, salts and cyclic derivatives thereof.

[0008] By “a modified lactoferrin peptide” is meant a peptide which isbased on lactoferrin or, more particularly, on a fragment thereof.Lactoferrin has been identified in a large number of organisms and thisinvention relates to modifications of lactoferrin and fragments thereoffrom all species. In particular, the invention relates to modifiedfragments of lactoferrin derived from mammals, especially modifiedfragments of bovine, murine, human, porcine and caprine lactoferrin.

[0009] LFB has been found to be more antimicrobially active than bovinelactoferrin itself and the peptides of the invention will preferably bemodified LFB or fragments thereof or the equivalent region to LFB inlactoferrin from other species.

[0010] The incorporation of an extra bulky and lipophilic amino acidinto a lactoferrin derived peptide is considered to be a modificationaccording to the invention.

[0011] The peptide will have 60% or more, preferably 70% or even 80%homology with the corresponding fragment of naturally occurringlactoferrin, e.g. of LFB or a fragment thereof.

[0012] For the purposes of the present invention, the term “sequencehomology” is not used to refer to sequence identity but to the presenceof either the same amino acid or one from the same functional group. Thestandard genetically coded amino acids can be grouped according to theircharacteristics, particularly of polarity and charge. Convenientgroupings are, glycine and alanine, serine, threonine, asparagine,glutamine and cysteine, lysine, arginine and histidine, aspartic acidand glutamic acid and valine, leucine, isoleucine, methionine,phenylalanine, tryptophan and tyrosine.

[0013] Of the 20 standard genetically coded amino acids, valine,leucine, isoleucine, methionine, tyrosine, tryptophan and phenylalanineare intended to be covered by the term “bulky and lipophilic aminoacid”, isoleucine, tyrosine, tryptophan and phenylalanine beingpreferred. In certain circumstances therefore, because even within thisgroup of bulky/lipophilic amino acids there is a gradual increase in thedegree of bulkiness/lipophilicity, an enhanced effect may be caused byreplacing one of the less bulky/lipophilic residues e.g. valine by oneof the more bulky/lipophilic residues such as tyrosine or tryptophan.These amino acids are conveniently referred to herein as “genetic” aminoacids and can be contrasted with a second group of amino acids which are“non-genetic”, i.e. which may be naturally occurring but are not codedfor by the 3 letter genetic code.

[0014] Throughout this specification, the widely used and understoodthree letter and one letter code for the 20 standard amino acids hasbeen used. Replacement of an amino acid from one group with anotheramino acid in the same group is conveniently referred to as a“conservative substitution”. Such substitutions do not generallymaterially effect the properties of the peptides of the invention andwhere any peptide differs from another only by such substitutions, ifone peptide is a peptide according to the present invention thentypically the other peptide will also be a peptide according to theinvention.

[0015] “Non-genetic” bulky and lipophilic amino acids include naturallyoccurring and synthetic amino acids having a bulky and lipophiliccharacter as well as genetic and non-genetic amino acids whose bulkinessand/or lipophilicity have been enhanced by chemical modification. A widevariety of amino acids and amino acid derivatives having a bulky andlipophilic character are known to the skilled man and are intended to beincluded within the term “bulky and lipophilic amino acid”.

[0016] The “extra” amino acid may refer to the addition or substitutionof a standard (genetic) bulky and lipophilic amino acid not present inthe native peptide, to an amino acid present in the native peptide whichhas been modified to make it more bulky and lipophilic or to theaddition or substitution of a non-genetic amino acid. When the extraamino acid is ‘added’, then all original amino acids in the peptideremain. When the extra amino acid is “substituted”, it replaces one ofthe naturally occurring amino acids. The bulky and/or lipophilic aminoacids are preferably present in place of other, naturally occurring,non-essential amino acids. By “non-essential” is meant an amino acidwhose presence is not required for the peptide as a whole to demonstratecytotoxic activity.

[0017] The different amino acids and their derivatives which fall withinthe definition of “bulky and lipophilic” amino acid may conveniently begrouped into three categories. The first group are those whose R group(a side chain), lends the amino acid a bulky and lipophilic character.The group of ‘genetic’ amino acids in this category has been definedearlier, for example, tryptophan has an indole R group and is considereda bulky and lipophilic amino acid. It has recently been found that oneof the standard protecting groups for arginine, PMC(2,2,5,7,8-penta-methylchroman-6-sulphonyl) can, under certainconditions, be transferred to tryptophan, resulting in sulphonation ofthe R group of tryptophan. Peptides wherein one or more tryptophanresidues have been modified in this way have surprising been shown tohave greatly enhanced cytotoxic activity. PMC modified tryptophan isthus a further example of an R group type bulky and lipophilic aminoacid.

[0018] It has surprisingly been found that amino acids or theirderivatives which have R groups of a certain size can be used to providemodified peptides which are particularly suitable for use as cytotoxicpeptides. Thus, according to a preferred aspect of the invention, theextra “bulky and lipophilic amino acid” is any amino acid or amino acidderivative, whose R group is uncharged and has at least 3, preferably 4or more, e.g. 7 or 8, more preferably at least 9 non-hydrogen atoms.Particularly preferred non-genetic bulky and lipophilic amino acids willhave at least 12, preferably at least 18 non-hydrogen atoms in the Rgroup. By way of example, the R group of the amino acid phenylalaninehas 7 non-hydrogen atoms and thus falls within our definition of “bulkyand lipophilic amino acids”.

[0019] Preferably, for ‘non-genetic’ amino acids, the R group in theextra bulky and lipophilic amino acid will have at least 8 or 9non-hydrogen e.g. carbon atoms, more preferably it should have at least2 closed rings of 5 or 6 atoms and conveniently these two rings arefused or bridged. The group may comprise only one ring which issubstituted by heavily branched alkyl groups, i.e. by groups whichinclude more than 1 branch site or 1 branch site which has attachmentsto 4 non-hydrogen atoms. The rings are formed of carbon atoms,optionally also including nitrogen, oxygen or sulphur atoms.Particularly preferred amino acids comprise a substituted orunsubstituted indole. The group should preferably be three-dimensional.Preferred non-genetic bulky and lipophilic amino acids includeadamantylalanine, 3-benzothienylalanine, 4,4′-biphenylalanine,3,3-diphenylalanine, homophenylalanine, 2,6-dichlorobenzyltyrosine,Cyclohexyltyrosine, 7-Benzyloxytryptophan, tri-tert-butyltryptophan,Homotryptophan, 3-(-Anthracenyl)-L-alanine, L-p-iso-propylphenylalanine,L-Thyroxine, 3,3′,5-Triiodo-L-thyronine.

[0020] Suitable bulky and lipophilic amino acid residues will thereforeinclude naturally occurring and non-naturally occurring amino acidswhich have an R group as previously defined, e.g. tryptophan oradamantylalanine or any amino acid, including genetically coded aminoacids, whose R groups have been modified to provide a bulky andlipophilic amino acid.

[0021] Bulky and lipophilic amino acids in this second category includemodified tryptophan and phenylalanine residues, in particular tryptophanresidues which have been substituted at the 1-, 2-, 5- and/or 7-positionof the indole ring, positions 1- or 2- being preferred.

[0022] A variety of other non-genetically coded and synthetic aminoacids and their derivatives having a bulky and lipophilic character areknown to the skilled man and are intended to be included within the term“bulky and lipophilic” amino acid. Suitable amino acids includethyroxine and the following commercially available amino acids and theirderivatives:

[0023] L-3-benzothienylalanine, CAS=72120-71-9 (Synthetech),D-3-benzothienylalanine, CAS=111139-55-0 (Synthetech),L-4,4′-biphenylalanine (Synthetech), D-4,4′-biphenylalanine(Synthetech), L-4-bromophenylalanine, CAS=24250-84-8 (Synthetech),D-4-bromophenylalanine, CAS=62561-74-4 (Synthetech),L-2-chlorophenylalanine, CAS=103616-89-3 (Synthetech),D-2-chlorophenylalanine, CAS=80126-50-7 (Synthetech),L-3-chlorophenylalanine, CAS =80126-51-8 (Synthetech),D-3-chlorophenylalanine, CAS=80126-52-9 (Synthetech),L-4-chlorophenylalanie, CAS=14173-39-8 (Synthetech),D-4-chlorophenylalanine, CAS=14091-08-8 (Synthetech),L-3-cyanophenylalanine, CAS=57213-48-6 (Synthetech),D-3-cyanophenylalanine (Synthetech), L-4-cyanophenylalanine(Synthetech), D-4-cyanophenylalanine (Synthetech),L-3,4-dichlorophenylalanine, CAS=52794-99-7 (Synthetech),D-3,4-dichlorophenylalanine, CAS=52794-98-6 (Synthetech),L-3,3-diphenylalanine (Synthetech), D-3,3-diphenylalanine (Synthetech),L-homophenylalanine, CAS=943-73-7 (Synthetech), D-homophenylalanine,CAS=82795-51-5 (Synthetech), L-2-indanylglycine (Synthetech),D-2-indanylglycine (Synthetech), L-4-iodophenylalanine, CAS=24250-85-9(Synthetech), D-4-iodophenylalanine, CAS=62561-75-5 (Synthetech),L-1-naphthylalanine, CAS=55516-54-6 (Synthetech), D-1-naphthylalanine,CAS=78306-92-0 (Synthetech), L-2-Naphthylalanine, CAS=58438-03-2(Synthetech), D-2-naphthylalanine, CAS=76985-09-6 (Synthetech),L-3-trifluoromethylphenylalanine, CAS=14464-68-7 (Synthetech),D-3-trifluoromethylphenyl-alanine (Synthetech),L-4-trifluoromethylphenylalanine, CAS=114926-38-4 (Synthetech),D-4-trifluoromethyl-phenylalanine, CAS=114872-99-0 (Synthetech),Boc-L-tert-leucine (Neosystem Laboratoire), Fmoc-L-tert-leucine(Neosystem Laboratoire), Fmoc-D-homoleucine (Neosystem Laboratoire),Fmoc-L-homoleucine (Neosystem Laboratoire), Boc-D-homophenylalanine(Neosystem Laboratoire), Boc-L-homophenylalanine (NeosystemLaboratoire), Fmoc-4-methyl-D-phenylalanine (Neosystem Laboratoire),Fmoc-4-methyl-L-phenylalanine (Neosystem Laboratoire),2,6-dichlorobenzyltyrosine, CAS =40298-71-3 (Senn Chemicals),Benzyltyrosine Fmoc (Senn Chemicals), Cyclohexyltyrosine Fmoc (SennChemicals), L-t-butylcysteine, CAS 2481-09-6 (Senn Chemicals),D-t-butylcysteine (Senn Chemicals), 1-Aminocyclopentane-1-carboxylicacid, CAS=52-52-8 (Senn Chemicals), L-3,5-diiodotyrosine, CAS=300-39-0(Senn Chemicals), D-3,5-diiodotyrosine (Senn Chemicals),L-3,5-dibromotyrosine (Senn Chemicals), D-3,5-dibromotyrosine (SennChemicals), L-t-butyltyrosine (Senn Chemicals), L-t-butyltyrosine (SennChemicals), N-Acetylhomotryptophan (Toronto Research),7-Benzyloxytryptophan (Toronto Research), Homotryptophan (TorontoResearch), 3-(-Anthracenyl)-L-alanine Boc (or Fmoc) (PeninsulaLaboratories), 3-(3,5-Dibromo-4-chlorophenyl)-L-alanine (PeninsulaLaboratories), 3-(3,5-Dibromo-4-chlorophenyl)-D-alanine (PeninsulaLaboratories), 3-(2-Quinoyl)-L-alanine Boc (or Fmoc) (PeninsulaLaboratories), 3-(2-Quinoyl)-D-alanine Boc (or Fmoc) (PeninsulaLaboratories), 2-Indanyl-L-glycine Boc (Peninsula Laboratories),2-Indanyl-D-glycine Boc (Peninsula Laboratories), Cyclopentyl-L-glycineBoc (Peninsula Laboratories), Cyclopentyl-D-glycine Boc (PeninsulaLaboratories), L-γ-Methyl-leucine Fmoc (Peninsula Laboratories),L-p-t-butoxyphenylglycine Fmoc (RSP), L-2-t-butoxyphenylalanine Fmoc(RSP), L-3-t-butoxyphenylalanine Fmoc (RSP), L-homotyrosine, O-t-butylether Fmoc (RSP), L-p-t-butoxymethylphenylalanine Fmoc (RSP),L-p-methylphenylalanine Fmoc (RSP), L-p-ethylphenylalanine Fmoc (RSP),L-p-iso-propylphenylalanine Fmoc (RSP), L-p-methoxyphenylalanine Fmoc(RSP), L-p(tBu-thio)phenylalanine Fmoc (RSP),L-p-(Trt-thiomethyl)phenylalanine Fmoc (RSP),L-p-hydroxymethyl-phenylalanine, O-t-butyl (RSP),L-p-benzoylphenylalanine (Advanced ChemTech), D-p-benzoyl-phenylalanine(Advanced ChemTech), L-α-cyclohexylglycine HCl (Advanced ChemTech),D-α-cyclohexylglycine HCl (Advanced ChemTech), O-benzyl-L-homoserine Boc(Advanced ChemTech), O-benzyl-D-homoserine Boc (Advanced ChemTech),L-β-1-Naphthyl-alanine (Advanced ChemTech), D-β-1-Naphthyl-alanine(Advanced ChemTech), L-penta-fluorophenylalanine Boc (AdvancedChemTech), D-penta-fluorophenylalanine Boc (Advanced ChemTech),D-penta-fluorophenylalanine Fmoc (Advanced ChemTech),3,5-Diiodo-L-tyrosine Fmoc (Boc) (Advanced ChemTech), L-Thyroxine Na,CAS=6106-07-6 (Novabiochem), 3,3′,5-Triiodo-L-thyronine Na, CAS=55-06-1(Novabiochem).

[0024] Surprisingly, it has been found that standard chemical protectinggroups when attached to an R group and thus increasing the bulk andlipophilicity of the residue can increase the bioactivity of peptides.Such protecting groups are well known in the art. Suitable protectinggroups which can significantly enhance anti-bacterial activity includePmc (2,2,5,7,8-pentamethylchroman-6-sulphonyl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulfonyl) and Pbf(2,2,4,6,7-pentamethyldihydrobenzofuransulfonyl) which may convenientlyincrease the bulk and lipophilicity of aromatic amino acids, e.g. Phe,Trp and Tyr. Also, the tert butyl group is a common protecting group fora wide range of amino acids and is capable of providing bulky andlipophilic amino acids as described herein, particularly when modifyingaromatic residues. The Z-group (carboxybenzyl) is a further protectinggroup which can be used to increase the bulky and lipophilicity of anamino acid to provide a peptide in accordance with the invention.

[0025] Although an initial observation of increased bioactivity was as aresult of a serendipitous transfer of the protecting group Pmc withinthe peptide from the guanidino group of arginine to tryptophan, aminoacids such as Trp which carry the protecting group can be synthesiseddirectly and incorporated into the peptide. This observation of thetransfer of Pmc from Arg to Trp has been observed by Stierandova et al.in Int. J. of Peptide Science (1994) 43, 31-38. Peptides in accordancewith the invention can be made by utilising this transfer of theprotecting group from Arg to Trp. When these two amino acids areseparated by 1-3 amino acids, the transfer of Pmc is most efficient.Peptides according to the invention may thus conveniently comprise anamino acid carrying a protecting group, e.g. Trp with Pmc attached inthe 2 position of the indole ring. The Pmc group may be attached to aTrp which has been added or to a Trp residue present in the originalpeptide. In a preferred embodiment of the invention, peptides willincorporate one or more additional tryptophan residues which can then bemodified to further increase its bulky and lipophilic character and thusprovide a peptide according to the invention.

[0026] Secondly, the bulky and lipophilic nature of an amino acid andthus of the peptide can also be enhanced by N- or C-terminalmodification and such modifications result in further peptides accordingto the present invention.

[0027] Thus peptides may, in addition to or instead of incorporating abulky and lipophilic R group, therefore be modified to incorporate abulky and lipophilic group at the N- and/or C-terminus. In this case,the N- or C-terminal residue which is modified to include a bulky andlipophilic N- or C-terminal group is the “extra bulky and lipophilicamino acid”. The bulky and lipophilic N- or C-terminal groups includeorganic groups such as protecting groups, especially Fmoc, Boc or otherstandard N terminal protecting groups or branched, linear or cyclicalkyl groups of formula CH₃(CH₂)_(n) wherein n is between 5 and 20,preferably between 8 and 14 and most preferably 10 to 12 or branched,linear or cyclic acyl groups having between 6 and 21, preferably 9 and15 and most preferably 11 to 13 carbon atoms.

[0028] Particularly, it has been found that peptides havingantibacterial and/or antitumor activity but a low toxicity can be madeby incorporating N-terminal modifications which include a cyclic group,preferably a 5- or 6-membered ring which may be alkyl or aryl, e.g.benzyl. More preferably the group which comprises the N-terminalmodification encompass 2 or more fused rings one or more of which may bea 5-membered ring e.g. adamantyl or Fmoc. It has surprisingly been foundthat groups which are three dimensional in character, such as thosewhich incorporate a fused ring system which do not lie in a single planehave particularly advantageous properties.

[0029] More specifically, it has been found that peptides havingantibacterial and/or antitumoral activity but a low toxicity can be madeby incorporating N-terminal modifications which include a cyclic group,preferably a 5- or 6-membered ring which may be alkyl or aryl. Morepreferably the group which comprises the N-terminal modificationencompasses 2 or more fused rings one or more of which may be a5-membered ring e.g. adamantyl or Fmoc. It has surprisingly been foundthat groups which are three dimensional in character, such as thosewhich incorporate a fused ring system which does not lie in a singleplane have particularly advantageous properties.

[0030] Suitable molecules which could be used to modify the N-terminusinclude:

[0031] cis-Bicyclo[3.3.0]octan-2-carboxylic acid, [18209-43-3](Aldrich); Abietic acid, [514-10-3] (Aldrich); Ursolic acid, [77-52-1](Aldrich); (1,2-Methanofullerene C₆₀)-61-carboxylic acid, [155116-19-1](Fluka); Dimethyl cubane-1,4-dicarboxylate, [29412-62-2] (Fluka);2-Norbornaneacetic acid, [1007-01-8] (Aldrich);4-Pentylbicyclo[2.2.2]octane-1-carboxylic acid, [73152-70-2] (Aldrich);3-Noradamantanecarboxylic acid, [16200-53-6] (Aldrich); 9-Fluoreneaceticacid, [6284-80-6] (Aldrich); cis-Decahydro-1-naphthol, [36159-47-4](Aldrich); 9-Ethyl-bicyclo[3.3.1]nonane-9-ol, [21915-33-3] (Aldrich);3-Quinuclidinol, [1619-34-7] (Aldrich); [[(1S)-endo]-(−)-Borneol,[464-45-9] (Aldrich); (1R,2R,3R,5S)-(−)-Isopinocampheol, [25465-65-0](Aldrich); Dehydroabietylamine [1446-61-3] (Aldrich);(±)-3-Aminoquinuclidine [6530-09-2] (Aldrich); (R)-(+)-Bornylamine,[32511-34-5] (Aldrich); 1,3,3-Trimethyl-6-aza-bicylo[3.2.1]octane[53460-46-1] (Aldrich); 1-Adamantylamine, [768-94-5] (Aldrich);9-Aminofluorene, [5978-75-6] (Aldrich); (1R)-(−)-10-Camphorsulfonicacid, [35963-20-3] (Aldrich); 5-Isoquinolinesulfonic acid, [27655-40-9](Aldrich); 2-Quinolinethiol, [2637-37-8] (Aldrich); 8-Mercaptomenthone,[38462-22-5] (Aldrich).

[0032] N-terminal modifications to provide peptides in accordance withthe invention will therefore typically comprise a bulky and lipophilicgroup R which may be attached directly to the N-terminal amine to form amono-, di- and possibly cationic trialkylated N-terminal amine.Alternatively, the R group may be attached via a linking moiety e.g. acarbonyl group (RCO) e.g. adamantyl or benzyl, carbamate (ROCO) e.g.Fmoc, or a linker which forms urea (RNHCO) or (R₂NCO) or by a linkerwhich forms a sulfonamide, boronamide or phosphonamide. Sulfonamideforming linkers may be particularly useful when a more stable peptide isrequired. The bulky and lipophilic group R comprises a preferablysaturated cyclic group, more preferably a polycyclic group wherein thecyclic groups are fused or bridged.

[0033] Peptides incorporating such N-terminal modifications areparticularly effective as anti-tumour peptides and surprisingly, thepresence of a cyclic, preferably multi-cyclic, N-terminal group providespeptides with an ability to kill tumour cells e.g. Meth A cells (from afibrosarcoma) but have little cytotoxic activity against normal cellse.g. red blood cells or normal fibroblast cells. For example,cyclohexyl—LFB 17-31 at a concentration of 46 μg/ml killed 50% of Meth Acells (murine sarcoma cell line) but did not kill 50% of red blood cellsor fibroblasts even at a concentration of 1000 μg/ml. This selectivityis, of course, highly desirable in the in vivo treatment of establishedtumours.

[0034] Particularly effective C-terminal modifications according to thepresent invention have also been investigated. Amidation of theC-terminus in order to manipulate the overall charge of a peptide isknown but it has now been found that larger C-terminal modifications,including the formation of esters, including thioesters or substitutedprimary and secondary amides result in peptides with good cytotoxicactivity. The C-terminal modifying groups will advantageously containmore than 4, preferably 6 or more non-hydrogen atoms and form e.g. abenzyl ester or amide. Other C-terminal groups include naphthylamine,substituted aromatic amines such as phenyl-ethylamine, mono, di- ortri-amino alkyl groups etc., groups incorporating a cyclic group beingpreferred. Standard C-terminal protecting groups are also available asactivity enhancing modification.

[0035] C-terminal modifications to provide peptides in accordance withthe invention will therefore typically comprise a bulky and lipophilicgroup R which may be attached directly to the C-terminal carboxy groupto form a ketone. Alternatively, the R group may be attached via alinking moiety, e.g. (OR) which forms an ester at the C-terminus, (NH—R)or (NR₂, wherein the two R groups needs not be the same) which formprimary and secondary amide groups respectively at the C-terminus orgroups (B—(OR)₂) which form boronic esters or phosphorous analogs. Thebulky and lipophilic group R preferably comprises at least 4non-hydrogen atoms.

[0036] Typically, the peptides of this aspect of the invention can berepresented by the following formula:

[0037] wherein X=a lactoferrin derived peptide of 7-25 amino acids inlength incorporating 3 cationic residues which is capable of forming anamphipathic α-helix;

[0038] R=SR¹, OR¹ or R¹; and

[0039] R=alkyl, cycloalkyl, aminoalkyl or aryl optionally substituted byhydroxy, alkoxy, acyloxy, alkoxycarbonyloxy, amino, oxo or fluoro groupsand optionally interrupted by oxygen, nitrogen, sulphur or phosphorousatoms.

[0040] The substituted R¹ groups may be mono or polysubstituted. Theterm “acyl” as used herein includes both carboxylate and carbonategroups.

[0041] As used herein, the term “alkyl” includes a long or short chainstraight-chained or branched aliphatic saturated or unsaturatedhydrocarbon group. R¹ may contain up to 40 non-hydrogen atoms,preferably between 4 and 12, more preferably 6 to 10 such atoms.

[0042] A lipophilic molecule is one which associates with its own kindin an aqueous solution, not necessarily because the interactions betweenthe lipophilic molecules are stronger than between the lipophilicmolecule and water but because interactions between a lipophilicmolecule and water would destroy the much stronger interactions betweenthe water molecules themselves. It is therefore preferable that thegroup which gives the amino acid its bulky and lipophilic character,whether it be an R group or N- or C-terminal modifying group, should notcontain many polar functional groups, e.g. no more than 4, preferably 2or less, such groups would increase the binding interaction with theaqueous surroundings and hence lower the lipophilicity of the molecules.Highly lipophilic groups thus being preferred. For example, a phenylgroup as a component of a bulky and lipophilic group would be preferredto a pyridyl group, even though they have the same number ofnon-hydrogen atoms and are of a similar overall size.

[0043] The bulky and lipophilic group would typically have at least 3 or4, preferably at least 5 or 6, more preferably 7 or more, mostpreferably 9 or more non-hydrogen atoms. The term ‘non-hydrogen’ atomsis used to indicate that hydrogen atoms are not included when countingthe number of atoms present in a group or molecule.

[0044] Peptides according to the invention may contain one or more ofthe above types of bulky and lipophilic groups, preferably the peptidewill incorporate at least one additional bulky and lipophilic R groupand either an N- or C-terminal modification as discussed above. Peptidesmay include all three types of bulky and lipophilic groups.

[0045] The peptides of the invention will typically contain between 1and 12, preferably 3 and 9, more preferably 4 to 7 bulky and lipophilicamino acids, depending on the length of the peptide. One or more, e.g. 1to 4 of these being extra bulky and lipophilic amino acids not presentin the native sequence. The ‘native sequence’ being the correspondingsequence (which may be identified using alignment software) in naturallyoccurring lactoferrin from any species. The native sequences typicallybeing a fragment of LFB or its equivalent in other species. The nativesequence for modification is selected and the modifications areidentified with reference to this sequence, e.g. LFB 17-31 W7 Pmc, a 15mer peptide having the sequence of residues 17-31 of bovine lactoferrinbut wherein the 7th residue has been replaced by tryptophan, one or moreof the tryptophan residues also being modified by Pmc. The nativesequence in terms of the order of amino acids is naturally occurring,although the particular fragment may be generated by chemical orenzymatic cleavage and not exist naturally at that length.

[0046] Sequence homology for such short peptides of the invention canmost simply be calculated by comparing the two sequences, residue forresidue, to determine whether the two amino acids at positions 1, 2, 3etc are the same or in the same group as previously defined. Thus, LFB(17-31)W3 has a 93.3% homology with LFB (17-31). Computer programs forcalculating sequence homology are also known in the art and these mayallow for additions (insertions) or deletions (gaps) in the sequence.

[0047] Amino acid sequence homology may be determined using the BestFitprogram of the Genetics Computer Group (CGC) Version 10 Software packagefrom the University of Wisconson. The program uses the local homologyalgorithm of Smith and Waterman with the default values: Gap creationpenalty=8, Gap extension penalty=2, Average match=2.912, Averagemismatch=−2.003. Such a program could therefore be used to assesshomology of the peptides of the invention with the native sequence,particularly if the modified peptide also incorporates gaps orinsertions. Such a program is most suited to establishing the alignmentbetween two sequences, again particularly when the modified sequenceincorporates gaps or insertions.

[0048] For such peptides which comprise only genetically coded aminoacids, similarity of the modified peptides with a known or naturalcytotoxic peptide can be expressed by stringency of hybridisation ofnucleic acid molecules encoding the two sequences rather than %homology. In this case, the ssDNA molecule encoding the modified peptideshould hybridise with the ssDNA molecule complementary to the ssDNamolecule which encodes the known or natural cytotoxic peptide.

[0049] Sequences which “hybridise” are those sequences binding(hybridising) under non-stringent conditions (e.g. 6× SSC, 50% formamideat room temperature) and washed under conditions of low stringency (e.g.2× SSC, room temperature, more preferably 2× SSC, 42° C.) or conditionsof higher stringency (e.g. 2× SSC, 65° C.) (where SSC=0.15M NaCl, 0.015Msodium citrate, pH 7.2).

[0050] Preferbly, the sequences will hybridise under conditions ofhigher stringency as defined above, or but for the degeneracy of thecode, the sequences would hybridise under high stringency conditions.

[0051] Preferably, the peptides will include 1 or 2 additional geneticbulky and lipophilic amino acids and may otherwise be identical to theknown cytotoxic peptide or incorporate only conservative substitutions.

[0052] Further preferred peptides according to the present inventioninclude those which have retained the original sequence of a fragment oflactoferrin (and could thus be considered to have 100% sequence homologywith the native sequence) but wherein the extra bulky and lipophilicamino acid is in the form of a modification to one of the originalresidues. This modification may be to the N- or C-terminus of thepeptide, in which case the N- or C-terminal residues would be the extrabulky and lipophilic amino acid or to the R goup of one of the residues.Preferred amongst such modifications is modifying an already bulky aminoacid such as tryptophan to make it more bulky, in the context of thepresent invention, this is still considered to be an “extra” bulky andlipophilic amino acid.

[0053] Peptides incorporating an extra bulky and lipophilic amino acidwill preferably exhibit an enhanced cytotoxic effect against bacterialor tumour cells while the toxicity of the peptides, e.g. their hemolyticactivity is reduced or only moderately increased as compared to thenative or original peptide.

[0054] In the context of the present invention, “cyclic derivatives”refers to peptides which are cyclic as a result of one or moredi-sulphide bridges. For some peptides incorporating two or cysteineresidues, this will be the naturally occurring form and production of alinear peptide will require the modification of the cysteine residues.

[0055] Typically, the peptide prior to incorporation of an extra bulkyand lipophilic amino acid will exhibit some cytotoxic activity, thisactivity being enhanced by the incorporation of a non-genetic bulky andlipophilic amino acid.

[0056] In absolute terms, the peptides according to the invention willincorporate a certain number of extra bulky and lipophilic amino acids.Typically between 1 and 6, preferably 1 and 4 extra bulky and lipophilicamino acids, depending on the overall length of the peptide. For examplea 7 to 10 mer peptide may have between 1 and 3 extra bulky andlipophilic residues, a 19-25 mer peptide may have between 1 and 5,preferably between 2 and 3 extra bulky and lipophilic residues.

[0057] Peptides according to the invention may conveniently comprise anadditional standard bulky and lipophilic amino acid, e.g. Trp, as wellas an amino acid carrying a protecting group, e.g. Trp with Pmc attachedin the 2 position of the indole ring. The Pmc group may be attached tothe Trp which has been added or to a naturally occurring Trp residue.

[0058] In addition to the bulky and lipophilic modifications, thelactoferrin derived peptides according to the invention mayadvantageously incorporate further modifications. In particular,increasing the overall positive charge of the peptide, for example byreplacing one or more naturally occurring amino acids, particularlynon-essential amino acids, with positively charged residues such aslysine or arginine may further enhance the activity of the lactoferrinpeptide. “Positively charged” refers to the side chain (R group) of theamino acid residue which has a net positive charge at pH 7.0. In thecase of peptides for use as anti-tumour agents, where the peptide mayadvantageously be capable of forming α-helix, substitutions within thepeptide sequence which serve to lower the angle subtended by thecationic sector, i.e. the angle of the positively charged face of thehelix may further enhance activity. In fact, lowering the anglesubtended may have a greater impact on activity than the net positivecharge per se. Other residues may advantageously be replaced by alamineresidues.

[0059] Peptides according to the invention may conveniently comprise anadditional bulky and lipophilic amino acid, e.g. Trp, as well as anamino acid carrying a protecting group, e.g. Trp with Pmc attached inthe 2 position of the indole ring. The Pmc group may be attached to theTrp which has been added or to a naturally occurring Trp residue.

[0060] For any lactoferrin derived peptide, suitable positions forincorporation of extra bulky and lipophilic amino acids in order toincrease cytotoxicity can be identified in a number of ways. Asdiscussed above, “incorporation” may include modification of an existingresidue. An alanine scan (involving sequential substitution of the aminoacids with alanine) can be used to identify non-essential amino acidswhich could be substituted by a bulky and lipophilic amino acid ormodified to increase its bulk and lipophilicity. Alternatively, acandidate peptide which forms an amphiphatic a-helix can be representedas a ‘helical wheel’ of residues and the cationic residues identified.These cationic residues will form positively charged domains or regionswithin the three-dimensional helical peptide structure and suitablepositions for incorporation of or modification to provide an extra bulkyand lipophilic amino acids are generally adjacent to or between suchcationic domains when viewed along the axis of the helical wheel.

[0061] It has even been found that peptides having enhancedantibacterial and/or antitumoural activity and preferably reducedtoxicity can be prepared by moving a bulky and lipophilic amino acidfrom its position in the original/native sequence to a region adjacentto the cationic sector, thus the oveall amino acid composition of thepeptide remains unchanged. Such 7-25 mer lactoferrin peptides which have3 or more cationic residues and are capable of forming an amphipathicα-helix and which have an extra bulky and lipophilic amino acid adjacentto the cationic sector, said extra bulky and lipophilic amino acid beingtaken from another, non-preferred, position in the sequence constitute afurther aspect of the present invention. In place of the bulky andlipophilic amino acid can be put the residue from adjacent to thecationic sector which the bulky and lipophilic amino acid replaces orany other less bulky and lipophilic amino acid. Suitable bulky andlipophilic amino acids in non-preferred positions which can be movedinto the region adjacent to the cationic sector (preferred position) canbe identified by e.g. an alanine scan which identifies non-essentialamino acids or by studying a helical wheel arrangement, non-preferredpositions typically being opposite a cationic domain.

[0062] It has also been found that peptides having reduced toxicity butstill having reasonable antibacterial or anti-tumoural activity (i.e.having enhanced selectivity) may be prepared by replacing anon-essential highly bulky and lipophilic amino acid such as tryptophanor phenylalanine with a less bulky and lipophilic amino acid e.g.isoleucine or leucine or even alanine or lysine. Generally, a“non-essential” bulky and lipophilic amino acid will be positioned onthe opposite side of the helix from the cationic sector, suchnon-essential bulky and lipophilic amino acids can be identified using ahelical wheel diagram or by an alanine scan. These peptides shouldnevertheless retain at least 3 bulky and lipophilic amino acids asherein defined. Thus, modified cytotoxic peptides having 7 to 25 aminoacids, at least three cationic residues and at least three bulky andlipophilic amino acids and being capable of forming an amphipathicα-helix, wherein one non-essential tryptophan or phenylalanine residuein the original/native sequence is replaced by a less bulky andlipophilic residue e.g. isoleucine or alanine constitute a furtheraspect of the present invention.

[0063] Other suitable sites for incorporation of a bulky and lipophilicamino acid are positions at or near, preferably adjacent, to an existinglipophilic amino acid. Proximity is judged in terms of the secondaryrather than primary structure of the peptide. The techniques involved inperforming an alanine scan and in constructing helical wheel diagramsare well known in the art.

[0064] In the case of LFB(17-31) (a 15 amino acid fragment of LFB whichlacks the ten C-terminal residues), non-essential amino acids determinedusing an alanine scan were Cys(3), Gln(7) and Gly(14), here thenumbering is in absolute terms relating to the peptide itself. Analogsof LFB(17-31) wherein these amino acids are replaced by bulky andlipophilic amino acids may be particularly effective.

[0065] Particularly preferred peptides according to the presentinvention are those which are based on bovine lactoferrin (LFB) orfragments (e.g. LFB 17-31) thereof or the equivalent fragment oflactoferrin from other animals.

[0066] A particular advantage of the peptides of the present inventionis their small size, peptides having 15 or fewer amino acids beingpreferred, conveniently of 9 or 10 amino acids or less. One sucheffective small peptide is LFB(17-27) wherein the Lys28, Leu29, Gly30and Ala3l from the C-terminal end of LFB(17-31) have been omitted. Thepeptides may be produced by any known method, conveniently by enzymaticdigestion or chemical cleavage of native peptides and subsequentmodification or by direct synthesis from the amino acid building blocks.The shorter the desired peptide the better as far as manufacture isconcerned, particularly for direct synthesis which is the preferredmethod of manufacture, as this limits the problems associated withchirality of the amino acids. In addition, short peptides are good forbiodelivery. There is a growing demand for antibiotics which can beadministered without the need for an injection, such as by inhalationand absorption across the blood capillaries of the nasal passages. A 10mer peptide could easily be administered in this way but peptides inexcess of 25 amino acids in length could not be delivered by inhalation.

[0067] It would also be desirable to increase the circulating half-lifeof the peptide and this could be achieved by further modifying thepeptides of the invention to include artificial amino acids as they areresistant to enzymatic breakdown. Long peptides are susceptible tobreakdown by endopeptidases which cleave internally of the peptide,shorter peptides would be less vulnerable to cleavage by endopeptidasesand breakdown by exopeptidases, which attack the ends of a peptide,could be reduced by acetylating the N terminus and otherwise blockingthe C terminus.

[0068] It has also been observed that the incorporation of enantio aminoacids can significantly increase the bioactivity of the peptides of theinvention and such peptides constitute a further preferred embodiment ofthe present invention. Excellent antimicrobial activity has been shownfor Enantio peptides which are the exact mirror image of the nativepeptide and Retro-Enantio peptides which adopt the same a-helicalconfirmation as the native peptide except the amide bonds point inopposite directions. Thus a further aspect of the present inventioncomprises the enantio or retro-enantio form of a lactoferrin derivedpeptide, particularly LFB or fragments thereof; as well as lactoferrinderived peptides which incorporate one or more D amino acids. Furtherpreferred embodiments of the present invention are thus peptides asdefined herein which incorporate one or more extra bulky and lipophilicamino acids and also comprise one or more D amino acids e.g. ⅓ or{fraction (3/2)} or ⅔ of the amino acids in the peptide are in the Dform and these may be arranged in any way throughout the sequence, e.g.alternately with L amino acids.

[0069] Enantio amino acids are also resistant to enzymatic breakdown andthe resultant increase in half-life of the peptides may go some way toexplaining the enhanced anti-bacterial activity. Enantio amino acids areexpensive and this is a further reason why the relatively short peptidesof the present invention are particularly advantageous.

[0070] By the term “capable” of forming an amphipathic α-helix is meantthat the peptide may, in certain circumstances, form an α-helix.Peptides may not necessarily have the α-helix as their naturalconfiguration in aqueous media but are able, for example in the presenceof helix providing substances such as Sodium dodecylsulphate (SDSS,2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP)or micelles (other than SDS) and cell membranes (artificial and natural)to form an a-helix or substantially α-helical structure. Circulardichroism may conveniently be used to test for the presence of anα-helix. Of more importance than the formation of an α-helix is the factthat the peptides are amphipathic, i.e. that the 20 structure of thepeptide, whether it is a a-helical or not is amphipathic. This isevidenced by the activity of the enantio peptides of the invention andpeptides incorporating D amino acids which are not a-helical.

[0071] In addition, the present invention relates to non-peptidecompounds showing the same cytotoxic activity as their proteinaceouscounterparts. Such petidomimetics or “small molecules” capable ofmimicking the activity of a protein or peptide are likely to be bettersuited for e.g. oral delivery due to their increased chemical stability.Such compounds will include a part which corresponds to the “extra bulkyand lipophilic amino acid” as previously defined. They will include agroup which corresponds to the R group, or N- or C-terminal modifyinggroup of said extra bulky and lipophilic amino acid.

[0072] It is now commonplace in the art to replace peptide orprotein-based active agents e.g. therapeutic peptides with suchpeptidomimetics having functionally-equivalent activity. Variousmolecular libraries and combinatorial chemistry techniques exist and areavailable to facilitate the identification, selection and/or synthesisof such compounds using standard techniques (Kieber-Emons, T. et al.Current Opinion in Biotechnology 1997 8: 435-441). Such standardtechniques may be used to obtain the peptidomimetic compounds accordingto the present invention, namely peptidomimetic organic compounds whichshow substantially similar or the same cytotoxic activity as thepeptides of the invention, e.g. as described herein in the Examples.

[0073] A further aspect of the invention thus provides a biomimeticorganic compound based on the peptides of the invention, characterisedin that said compound exhibits cytotoxic, e.g. antibacterial orantitumoural activity, at at least the level exhibited by the peptidesof the invention as hereinbefore defined.

[0074] The term “cytotoxic” is intended to refer not only to an activityagainst prokaryotic cells but also against eukaryotic cells. Although incertain circumstances it is desirous to have a peptide which has a goodanti-bacterial activity but does not lyse or otherwise destroy the cellsof the patient, peptides within the scope of the present invention havebeen shown to have an anti-tumoural activity. The anti-tumoural activityof these peptides and medicaments containing them constitute furtheraspects of the present invention. Anti-tumoural activity includes thedestruction or reduction in size or number of benign or malignanttumours and the prevention or reduction of metastasis.

[0075] In general, lactoferrin derived peptides according to theinvention which have no non-genetic amino acids and have a good activityagainst tumour cells will have 25-10, preferably 12-20 e.g. 18 aminoacids. Peptides according to a non genetic bulky and lipophilic groupand having good anti-tumoural activity will generally be shorter, with7-20, preferably 10-20, more preferably 10-15 amino acids. By way ofexample, LFB 17-27 A7, M3, R2, 11W4,10, Y1-NH₂ PMC and LFB 18-24 R1,7W2,3,6-NH₂ PMC require only 50 and 38 μg/ml respectively to kill 50% ofMeth A cells.

[0076] In general, peptides having good activity against tumours will belonger than those exhibiting good anti-bacterial activity.Anti-bacterial peptides will typically have 7 to 20, preferably 7 to 14,e.g. 8 or 9 amino acids.

[0077] The anti-tumoural activity of the modified peptides is muchbetter than could be predicted merely from the fact that the peptidesappear to have a lytic effect on bacterial cells. The observed lyticeffect on tumour cells in vitro is powerful and tumour regression inmice is very rapid, occurring within 3-6 days. It appears that there isinduction of an immunological memory, as inoculation of tumour cells inmice after the treatment and regression of the original tumour did notgive rise to any secondary tumour growth.

[0078] Importantly, we have demonstrated regression of establishedtumours, even with unmodified LFE. In this context, “unmodified” refersalso to fragments of LFB which exhibit this antitumoural activity, e.g.LFB(17-31). The peptide may be cyclic or linear, preferably cyclic. Thisnew clinical use of LFB represents a further aspect of the presentinvention as we provide for the use of LFB and fragments thereof in themanufacture of a medicament for the treatment of solid tumours. Theability to treat solid tumours is particularly useful when a tumour isunresectable. A further advantage is that the observed cytolytic effectin tumours is not species specific and thus the peptides have utility intreating human tumours.

[0079] In a further aspect of the present invention, we provide a methodof treating solid tumours which comprises administration of LFB or afragment thereof in a dose sufficient to cause regression of the tumour.

[0080] Suitable doses for treatment of tumours with bioactive peptideswill be known to the skilled man and doses used in the animalexperiments described herein can be used to estimate an appropriate dosefor other animal and human patients. Administration of a peptide may bedaily, more usually on alternate days or on every 3rd or 4th day. 1 to10, typically 2 to 5 administrations may result in successful treatment.Similar treatment protocols will be used for treatment of bacterial orviral infections.

[0081] Peptides according to the invention will preferably be at leastas cytotoxic as LFB (17-31). Some peptides according to the inventionwill be more active in some respects (e.g. antitumoural) than LFB(17-31) but less active in other respects e.g. against E. coli. Somepeptides may be less active but other properties e.g. a low hemolyticactivity will render them useful in certain applications.

[0082] It has also been observed that in certain circumstances, reducingthe bulk and lipophilicity of an amino acid may result in a significantdecrease in toxicity and only a marginal decrease in anti-bacterial oranti-tumoural activity, thus resulting in a very useful therapeuticpeptide. Specifically, replacing a tryptophan residue, the most bulkyand lipophilic of the genetic amino acids or phenylalanine with another,less bulky and lipophilic amino acid, e.g. isoleucine or leucine or byalanine or even lysine can result in peptides with good to moderateanti-bacterial or anti-tumoural activity and very low toxicity. Thesepeptides, wherein a tryptophan or phenylalanine residue has beenreplaced should nevertheless retain at least 3 bulky and lipophilicamino acids as herein defined. Thus, modified cytotoxic lactoferrinderived peptides having 7 to 25 amino acids, at least three cationicresidues and at least three bulky and lipophilic amino acids and whichis capable of forming an amphipathic α-helix, wherein a tryptophan orphenylalanine residue in the native sequence is replaced by a less bulkyand lipophilic residue e.g. isoleucine constitute a further aspect ofthe present invention. The replaced tryptophan or phenylalanine residueshould be non-essential and will typically be opposite a cationic sectorof the helix. Non-essential residues can be determined by an alaninescan or helical wheel drawing.

[0083] The antibacterial activity of the peptides of the invention maymanifest itself in a number of different ways. Certain modifications mayresult in peptides which are bacteriostatic and others in peptides whichare bacteriocidal. Advantageously, the majority of peptides according tothe invention are bacteriocidal. Thus, inter alia, the invention alsoprovides a method of inhibiting the growth of bacteria comprisingcontacting the bacteria with an inhibiting effective amount of acytotoxic peptide according to the invention.

[0084] The term “contacting” refers to exposing the bacteria to apeptide so that it can effectively inhibit, kill or lyse bacteria, bindendotoxin (LPS), or, permeabilize gram-negative bacterial outermembranes. Contacting may be in vitro, for example by adding the peptideto a bacterial culture to test for susceptibility of the bacteria to thepeptide. Contacting may be in vivo, for example administering thepeptide to a subject with a bacterial disorder, such as septic shock.“Inhibiting” or “inhibiting effective amount” refers to the amount ofpeptide which is required to cause a bacteriastatic or bacteriacidaleffect. Examples of bacteria which may be inhibited include E. coli, P.aeruginosa, E. cloacae, S. typhimurium and S. aureus. The method ofinhibiting the growth of bacteria may further include the addition ofantibiotics for combination or synergistic therapy. The appropriateantibiotic administered will typically depend on the susceptibility ofthe bacteria such as whether the bacteria is gram negative or grampositive, and will be easily discernable by one of skill in the art.

[0085] In addition, different modifications may enhance theantibacterial activity against certain types of bacteria more thanagainst other types. For example S. aureus is particularly susceptibleto very large bulky and lipophilic groups, typically those having atleast 12 or 18 non-hydrogen atoms in the R group e.g. those peptideswhich incorporate a Pmc modified tryptophan residue. In addition, Rgroups which are substantially planar have good activity against E.coli, while a more 3-dimensional group of comparable lipophilicity ispreferred for producing good activity against S. aureus.

[0086] It has been found that the sequence of bovine lactoferricin (LFB17-41) can be reduced by up to about 10 residues at the C-terminal end,e.g. to LFB(17-31) without significant loss of antibacterial activity.LFB 17-31=FKCRRWQWRMKKLGA. As well as bovine lactoferricins, we haveidentified the regions corresponding to LFB 17-31 in man,LFH=TKCFQWQRNMRKVRG, goat, LFC=SKCYQWQRRMRKLGA, mice,LFM=EKCLRWQNEMRKVGG and pigs, LFP=SKCRQWQSKIRRTNP and such regions arealso suitable for manipulation according to the invention.

[0087] A variant of the effects of an increase in lipophilicity ofcertain peptides discussed above has been observed and a further aspectof the present invention comprises a cytotoxic lactoferrin peptide of 15amino acids or less characterised in that it has an additionalbulky/lipophilic group at one end. In respect of this aspect of theinvention, the bulky/lipophilic group includes organic groups such asprotecting groups, especially Fmoc, Boc or other standard N terminalprotecting groups or branched, linear or cyclic alkyl groups of formulaCH₃(CH₂) wherein n is between 5 and 20, preferably between 8 and 14 andmost preferably 10 to 12 or branched, linear or cyclic acyl groupshaving between 6 and 21, preferably 9 and 15 and most preferably 11 to13 carbon atoms. For example, an LFB(17-31) peptide having aCH₃(CH₂)_(n) alkyl group at the N-terminal end had an up to 10 foldincrease in antibacterial activity. The groups are attached to N- orC-terminal or close, preferably adjacent, to N- or C-terminal residues.These groups may be attached to native amino acid residues, ornon-native amino acids carrying the bulky/lipophilic group may beincorporated into the peptide.

[0088] A still further aspect of the present invention is a method ofpreparing a peptide having enhanced cytotoxic activity and/or improvedselectivity for target cell types which comprises incorporating a bulkyand lipophilic amino acid into a 7 to 25 mer lactoferrin peptide withthree or more cationic residues which is optionally capable of formingan amphiphatic α-helix. Such a method would include, as well as addingor substituting a genetic or non-genetic bulky and lipophilic aminoacid, enhancing the bulky and lipophilic nature of one or more of theamino acids by modification of the R group or the N- or C-terminus.

[0089] “Incorporating” may include modification of an existing residueor introduction of such a residue into the peptide by addition orsubstitution, preferably substitution. A synthetic method may be usedwhereby the non-genetic bulky and lipophilic amino acid is included insequence in the growing peptide so no post peptide formation processingis required.

[0090] When, herein, we refer to a peptide having “enhanced” cytotoxicactivity, it is meant that the peptide which has been modified inaccordance with the invention has enhanced cytotoxicity against one ormore strains of bacteria or types of cancerous cells as compared to thepeptide without said modification. By “improved selectivity for targetcell types” is meant that the ratio of cytotoxic activity against targetcells as compared to non target cell types is increased. In other words,selectivity can be improved if, for example, the antibacterial activityof a peptide is the same before and after modification but the hemolyticactivity is decreased after modification. Similarly, useful peptidesaccording to the invention may be made even when hemolytic activityincreases, if the antibacterial or antitumoural activity increases by agreater amount. Selectivity may also refer to one type of bacteria overanother.

[0091] Tryptophan rich analogs of lactoferrin have been shown to beeffective as antimicrobial agents. Such analogs preferably have one ortwo tryptophan residues replacing other, non-essential residues.

[0092] In a further aspect, the invention provides a method of enhancingthe cytotoxic activity of a lactoferrin peptide of 7 to 25 amino acidsin length, which has three or more cationic residues and is optionallycapable of forming an amphipathic a-helix which comprises introducing byaddition or substitution, preferably substitution, an extra bulky andlipophilic amino acid (e.g. tryptophan).

[0093] A ‘lactoferrin peptide’ is a peptide derived from naturallyoccurring lactoferrin from any species. The peptides itself may not benaturally occurring, being a fragment of lactoferrin e.g. a fragment ofLFB.

[0094] The peptides of the invention may be directly synthesised in anyconvenient way. Generally the reactive groups present (for exampleamino, thiol and/or carboxyl) will be protected during overallsynthesis. The final step in the synthesis will thus be the deprotectionof a protected derivative of the invention. As discussed above, certainpeptides of the invention will carry a ‘protecting group’ as this isresponsible for enhanced cytotoxicity.

[0095] In building up the peptide, one can in principle start either atthe C-terminal or the N-terminal although the C-terminal startingprocedure is preferred. The non-genetic amino acid can be incorporatedat this stage as the sequence is extended or as a result of apost-synthetic modification.

[0096] Methods of peptide synthesis are well known in the art but forthe present invention it may be particularly convenient to carry out thesynthesis on a solid phase support, such supports being well known inthe art.

[0097] A wide choice of protecting groups for amino acids are known andsuitable amine protecting groups may include carbobenzoxy (alsodesignated Z) t-butoxycarbonyl (also designated Boc),4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will beappreciated that when the peptide is built up from the C-terminal end,an amine-protecting group will be present on the α-amino group of eachnew residue added and will need to be removed selectively prior to thenext coupling step.

[0098] Carboxyl protecting groups which may, for example be employedinclude readily cleaved ester groups such as benzyl (Bzl), p-nitrobenzyl(ONb), pentachlorophenyl (oPClP), pentafluorophenyl (OPfp) or t-butyl(OtBu) groups as well as the coupling groups on solid supports, forexample methyl groups linked to polystyrene.

[0099] Thiol protecting groups include p-methoxybenzyl (Mob), trityl(Trt) and acetamidomethyl (Acm).

[0100] A wide range of procedures exists for removing amine- andcarboxyl-protecting groups. These must, however, be consistent with thesynthetic strategy employed. The side chain protecting groups must bestable to the conditions used to remove the temporary α-amino protectinggroup prior to the next coupling step.

[0101] Amine protecting groups such as Boc and carboxyl protectinggroups such as tBu may be removed simultaneously by acid treatment, forexample with trifluoroacetic acid. Thiol protecting groups such as Trtmay be removed selectively using an oxidation agent such as iodine.

[0102] Peptides according to the invention may be prepared by incompletedeprotection to leave groups which enhance the cytotoxic activity of thepeptides. Alternatively, modified R and N- and C-terminal groups may beprepared after synthesis of the peptide and associated deprotection.

[0103] A particularly preferred method involves synthesis using aminoacid derivatives of the following formula: Fmoc-amino acid-Opfp.

[0104] A proportion of the peptides of the invention, i.e. those whereinthe extra bulky and lipophilic amino acid is one of the geneticallycoded amino acids will be capable of being expressed in prokaryotic andeukaryotic hosts by expression systems well known to the man skilled inthe art. Methods for the isolation and purification of e.g. microbiallyexpressed peptides are also well known. Polynucleotides which encodethese peptides of the invention constitute further aspects of thepresent invention. As used herein, “polynucleotide” refers to a polymerof deoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct, e.g. an expressionvector such as a plasmid. Polynucleotide sequences of the inventioninclude DNA, RNA and cDNA sequences. Due to the degeneracy of thegenetic code, of course more than one polynucleotide is capable ofencoding a particular peptide according to the invention.

[0105] When a bacterial host is chosen for expression of a peptide, itmay be necessary to take steps to protect the host from the expressedanti-bacterial peptide. Such techniques are known in the art and includethe use of a bacterial strain which is resistant to the particularpeptide being expressed or the expression of a fusion peptide withsections at one or both ends which disable the antibiotic activity ofthe peptide according to the invention. In the latter case, the peptidecan be cleaved after harvesting to produce the active peptide. If thepeptide incorporates a chemical modification then the activity of theexpressed peptide may be low, only enhanced to really cytotoxic levelsby post-synthetic chemical modification e.g. addition of Pmc.

[0106] The present invention also provides pharmaceutical compositionscontaining the peptides of the invention as defined above together withphysiologically acceptable excipients. Suitable diluents and carriersare known to the skilled man. The peptides of the invention for use inmethods of treatment, particularly in the treatment of prevention ofbacterial infections or as an anti-tumour agent (both in the destructionor reduction in size or number of benign or malignant tumours, which maybe ascites and in the prevention of metastasis) constitute furtheraspects of the present invention.

[0107] The compositions according to the invention may be presented, forexample, in a form suitable for oral, nasal, parenteral, intravenal,intratumoral or rectal administration.

[0108] As used herein, the term “pharmaceutical” includes veterinaryapplications of the invention.

[0109] The compounds according to the invention may be presented in theconventional pharmacological forms of administration, such as tablets,coated tablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. The peptides of the invention areparticularly suitable for topical administration, e.g. in the treatmentof diabetic ulcers. Conventional pharmaceutical excipients as well asthe usual methods of production may be employed for the preparation ofthese forms. Tablets may be produced, for example, by mixing the activeingredient or ingredients with known excipients, such as for examplewith diluents, such as calcium carbonate, calcium phosphate or lactose,disintegrants such as corn starch or alginic acid, binders such asstarch or gelatin, lubricants such as magnesium stearate or talcum,and/or agents for obtaining sustained release, such ascarboxypolymethylene, carboxymethyl cellulose, cellulose acetatephthalate, or polyvinylacetate.

[0110] The tablets may if desired consist of several layers. Coatedtablets may be produced by coating cores, obtained in a similar mannerto the tablets, with agents commonly used for tablet coatings, forexample, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titaniumdioxide or sugar. In order to obtain sustained release or to avoidincompatibilities, the core may consist of several layers too. Thetablet-coat may also consist of several layers in order to obtainsustained release, in which case the excipients mentioned above fortablets may be used.

[0111] Organ specific carrier systems may also be used.

[0112] Injection solutions may, for example, be produced in theconventional manner, such as by the addition of preservation agents,such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutionsare then filled into injection vials or ampoules.

[0113] Nasal sprays which are a preferred method of administration maybe formulated similarly in aqueous solution and packed into spraycontainers either with an aerosol propellant or provided with means formanual compression. Capsules containing one or several activeingredients may be produced, for example, by mixing the activeingredients with inert carriers, such as lactose or sorbitol, andfilling the mixture into gelatin capsules.

[0114] Suitable suppositories may, for example, be produced by mixingthe active ingredient or active ingredient combinations with theconventional carriers envisaged for this purpose, such as natural fatsor polyethyleneglycol or derivatives thereof.

[0115] Dosage units containing the compounds of this inventionpreferably contain 0.1-10 mg, for example 1-5 mg of the peptides of theinvention. The pharmaceutical compositions may additionally comprisefurther active ingredients, including other cytotoxic agents such asother antimicrobial peptides. Other active ingredients may includedifferent types of antibiotics, cytokines e.g. IFN-γ, TNF, CSF andgrowth factors, immunomodulators, chemotherapeutics e.g. cisplatin orantibodies.

[0116] A yet further aspect of the present invention provides thetherapeutic use of the peptides of the invention as defined above i.e.the peptides for use as medicaments, e.g. antibacterians or antitumouralagents. Further aspects comprise a method of treating or preventingbacterial infections in a patient comprising the administration to saidpatient of one or more of the peptides of the invention and a method oftreating tumours in a patient comprising the administration of one ormore of the peptides of the invention. The treatment of tumours includesthe destruction or reduction in size or number of benign or malignanttumours which may be ascites and the preventionn of metastasis.

[0117] A still further aspect of the present invention comprises the useof one or more of the peptides of the invention in the manufacture of amedicament for treating bacterial infections or tumours.

[0118] Anti-bacterial agents such as the peptides of the presentinvention have a wide variety of applications other than aspharmaceuticals. They can be used, for example, as sterilising agentsfor materials susceptible to microbial contamination. The peptides ofthe invention exhibit broad antimicrobial and antibiotic activity andthus are also suitable as anti-viral and anti-fungal agents which willhave pharmaceutical and agricultural applications and as promoters ofwound healing or spermicides. All of these uses constitute furtheraspects of the invention.

[0119] The peptides, when used in topical compositions, are generallypresent in an amount of at least 0.1%, by weight. In most cases, it isnot necessary to employ the peptide in an amount greater than 1.0%, byweight.

[0120] Anti-tumour peptides may be administered in combination, possiblyin synergistic combination with other active agents or forms of therapy,for example administration of a peptide according to the invention maybe combined with chemotherapy, immunotherapy, surgery, radiation therapyor with the administration of other anti-tumour peptides.

[0121] In employing such compositions systemically (intra-muscular,intravenous, intraperitoneal), the active peptide is present in anamount to achieve a serum level of the peptide of at least about 5ug/ml. In general, the serum level of peptide need not exceed 500 ug/ml.A preferred serum level is about 100 ug/ml. Such serum levels may beachieved by incorporating the peptide in a composition to beadministered systemically at a dose of from 1 to about 10 mg/kg. Ingeneral, the peptide(s) need not be administered at a dose exceeding 100mg/kg.

[0122] Those peptides exemplified herein represent preferred peptidesaccording to the invention. Any peptide whose specific sequence isdisclosed herein, particularly those peptides which are more activeagainst bacterial cells than LFB 17-31, constitute a further aspect ofthe present invention.

[0123] Some of the preferred non-genetic bulky and lipophilic aminoacids incorporated into the peptides of the invention includesubstituted tryptophans which provide an increase in bulk andlipophilicity and a significant increase in bioactivity. Substitutionshave been made at the 1-position (or the indole N-position) and theadjacent 2-position and these new compounds, described in Example 2constitute a still further aspect of the present invention. New1-substituted tryptophans include 1-benzyl and 1-tosyl tryptophan.

[0124] The following novel, 2-substituted Tryptophan residues have beenmade, Z-Trp (2-nitrophenylsulfenylchloride)-OH and oxides thereof andZ-Trp(2-Pmc)-OH wherein Z is a protecting group, e.g. Fmoc. Method II ofExample 2E is a newly devised synthetic route suitable for thepreparation of a range of 2-sulfones and constitutes a further aspect ofthe present invention. Therefore, we further provide a method ofpreparing tryptophan residues substituted at the 2-position of theindole ring which comprises transferring the group with which thetryptophan will be substituted from a guanidyl containing group to anN-protected tryptophan. Preferably the guanidyl containing group is anarylalkyl or alkyl guanidyl group, most preferably it is aphenylethylguanidyl group. Preferably the N-protecting group is Fmoc andpreferably the tryptophan substituting group is Pmc.

[0125] LFB 17-41 whose cysteine residues have been blocked bypyridylethylation or acetamido-methylation but incorporate no furtherbulky and lipophilic amino acids are not per se peptides of theinvention. However pharmaceutical compositions comprising these peptidesas well as use of the peptides as therapeutic agents as herein describedconstitute further aspects of the present invention.

[0126] The invention will now be described with reference to thefollowing non-limiting examples in which.

[0127]FIG. 1 shows the amino acid sequence and charge at pH 7 forsynthetic lactoferricins from different species;

[0128]FIG. 2 shows the effects of linear and cyclic lactoferricin B on aMeth A fibrosarcoma cell line in vitro after 24 hours incubation;

[0129]FIG. 3 shows the effects of different LFB derivatives on Meth Acells in vitro after ½ hour incubation, +=pmc-modified; −=unmodified;

[0130]FIG. 4 shows the effects of different LFB derivatives on Meth Acells in vitro after 4 hours incubation, +=pmc-modified; −=unmodified;

[0131]FIG. 5 shows the effects of pmc modified retro LFB 17-31(+), FmocLFB 17-31(A8) and LFB 17-31 on Meth A cells in vitro after 4 hour. RPMIwas used as negative control and Triton 100× as positive control.Concentrations are in mg/ml;

[0132]FIG. 6 shows the effects of pmc modified retro LFB 17-31(+), FmocLFB 17-31(A8) and LFB 17-31 on Meth A cells in vitro after 4 hours. RPMIwas used as negative control and Triton 100× as positive control.Concentrations are in mg/ml;

[0133]FIG. 7 shows the dose response on human promyelotic leukemia cellline HL 60 after 4 hours. HL 60 cells, 1×10⁴ were incubated withpeptides 50, 30, 20, 10, 5, 1 μg, 1000-20 μg/ml in 2 hours and colouredwith MTT;

[0134]FIG. 8 shows inhibition of tumor growth; Meth A tumor cells (5×10⁷cells) were inoculated on day 1 and treated on day 7 and day 10 with 0.5mg (1 mg of P1) of the different peptides;

[0135]FIG. 9 shows the effect of D-LFB (17-31) A7 Pmc-NH₂ on B16F10murine melanoma;

[0136]FIG. 10 shows the size of tumours established in Balb/c mice whoare reinoculated with Meth A cells after successful treatment with cLFB.The mice were not treated with cLFB or other peptides in the study, thussome form of adoptive immunity is shown. Reinoculation of Meth A cells 1month after the LFB-treatment of Meth A tumours.

EXAMPLE 1

[0137] Human, Bovine, Murine and Caprine Lactoferrin Derived Peptides

[0138] A) MIC (Minimum Inhibitory Concentration) Tests

[0139] The bacterial strains used were: Escherichia coli ATCC 25922 andStaphylococcus aureus ATCC 25923. All strains were stored at −70° C. Thebacteria were grown in 2% Bacto Peptone water (Difco 1807-17-4). Alltests were performed with bacteria in mid-logarithmic growth phase.Determination of the minimum inhibitory concentration (MIC) of thepeptides for bacterial strains were performed in 1% Bacto Peptone water.A standard microdilution technique with an inoculum of 2×10⁶ CFU/ml wasused. All assays were performed in triplets. Since the peptides arepositively charged and therefore could adhere to the plastic wells, wecontrolled the actual concentration of the peptides in the solution byHPLC. There was no difference between the concentration of the peptidesbefore or after adding the solution to the plastic wells.

[0140] B) Synthesis of Peptides

[0141] Initially, the lactoferricin B used was a gift from Wayne Bellamy(Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd, Japan).Later in the study the peptides were synthesised with a 9050 PlusPepSynthesizer (Milligen). All peptides were synthesised on solid phaseby use of fluorenylmethoxycarbonyl (Fmoc) chemistry. Cysteines incystein containing peptides were protected with acetamidomethyl groupsto prevent disulfide bridge formation. The peptides were analysed andpurified by reversed phase HPLC on a Waters 600E chromatograph(Millipore) with UV detection at 254 nm. The fractions purified on HPLCwere analysed on a liquid chromatography-mass spectrometer (LC-MS) withelectrospray interface (Fisons VG Quattro) or/and with Fast AtomBombardment Mass Spectrometry (FAB-MS) (Fisons VG Tribrid).

[0142] Structure of the Lactoferricins

[0143] The structure of human lactoferrin is determined to 2.8 and 2.2 Åresolution by X-ray crystallography. Human lactoferricin (LFH) consistsof residues 1-47 of human lactoferricin. LFH contains two peptidefragments; one consisting of residues 12-47 cyclised with a disulfidebridge between Cys20 and Cys37, the second fragment (residues 1-11) isconnected to the 12-47 fragment through a disulfide bridge between Cys10and Cys46. In the human lactoferrin structure, the correspondingresidues comprises a β-strand (residues 4-11), an α-helix (residues12-29), a turn (residues 30 and 31), followed by a β-strand (residues31-47) [Day, C. L., Anderson, B. F., Tweedie, J. W. and Baker, E. N.(1993) J. Mol. Biol. 232, 1084-1100]. Bovine lactoferricin (LFB) withonly 25 residues (residues 17-41) in a single chain is structurally muchsimpler than LFH.

[0144] Antibiotic Activity of Synthetic Lactoferricins with SequencesFrom Different Species

[0145] The amino acid sequence of lactoferrins from goat [Provost, F.L., Nocart, M., Guerin, G. and Martin, P. (1994) Biochem. Biophys. Res.Commun. 203, 1324-1332] and mouse [Pentecost, B. T. and Teng, C. T.(1987) J. Biol. Chem. 262 10134-10139] have been determined and showhigh sequence homology with both the human and the bovine lactoferrins.The residues engaged in the helix-turn-strand motif can easily beidentified in the sequence as shown in FIG. 1. As LFB is moreantibacterial than LFH, the residues corresponding to LFB (17-41) werechosen in the amino acid sequence of human, murine and caprinelactoferrin to prepare analogous lactoferricin peptides; LFH (18-42),LFM (17-41) and LFC (17-41) respectively. The disulfide bridge is notessential for antibiotic activity in bovine and human lactoferricin[Bellamy et al. (1992)] and all peptides were prepared with ACMprotection of the cysteine residues to avoid cyclisation or oxidation.

[0146] The antibacterial activities of the synthetic lactoferricinsexpressed as MIC are compiled in Table 1 which shows that LFB (17-41)displayed the most significant antibacterial activity against E. coliand S. Aureus. TABLE 1 Minimum inhibitory concentration (MIC) in μg/ml(μM) of synthetic lactoferricins on E. coli ATCC 25922 and S. aureusATCC 25923. E. coli S. aureus ATCC 25922 ATCC 25923 Peptide MIC MIC LFH(18-42) >200 >200 LFB (17-41)    30  30 LFM (17-41) >200 >200 LFC(17-31)   750 1000 LFB (14-31)    70 (28)  200 (80) LFB (17-31)    40(20)  100 (50) LFB (18-31)    80 (43)  200 (108) LFB (19-31)   200(120) >250 (150) LFB (20-31)   100 (62)  200 (124) LFB (17-31) K17    60(30)  100 (50) LFB (17-31) F20    20 (10)  200 (100) LFB (17-31)    20(10)  200 (100) K17, F20

[0147] A property considered to be important in determining theantibacterial activity of linear peptides, is their ability to adopthelical structures. In the intact lactoferrin protein, residues 14-28are located in an α-helix, residues 29-31 comprise a turn and residues32-41 are in a β-strand. We therefore anticipated that the antibacterialeffect of the lactoferricins could originate from the part of thesequence that participates in the helix of the intact protein. As thebovine lactoferricin sequence, LFB (17-41), was the only peptide withsignificant antibacterial property, we chose to prepare a shortervariety of the bovine peptide, LFB (17-31), containing both the helixand turn residues of the protein, while the 10 residues encompassing thestrand were removed. Despite the fact that LFB (17-31) has a lower netcharge (FIG. 1) than LFB (17-41) and LFC (17-41), it still retains mostof the antibacterial effect as shown in Table 1. These findings indicatethat even if the overall charge is important, it is not sufficient forantibacterial activity.

EXAMPLE 2

[0148] Preparation of Novel Substituted Tryptophans

[0149] In the following Examples and throughout the text the followinggeneral formula: Z-XX(n-y)-OH refers to a substituted amino acid (XX)wherein the NH₂ group of the amino acid is Z-protected, the amino acidis y-substituted at the n position and the COOH group of the amino acidis free.

[0150] A) Preparation of Ac-Trp(1-Tos)-OH

[0151] Experimental:

[0152] A mixture of Ac-Trp-OEt (0.199, 0.69 mmol), tosyl chloride(0.209, 1.04 mmol), tetrabutylammonium hydrogensulfate (2 mg, 0.01equiv.) and NaOH (0.079, 1.73 mmol) in dichloromethane was stirred atroom temperature for 2.5 hours. To the reaction mixture was addeddiluted HCl until a pH of 2-3 was reached and then washed with water. Tothe organic phase was added a diluted base and the aqueous phase wasextracted with dichloromethane, acidified and again extracted withdichloromethane.

[0153]¹H NMR (CDCl₃): δ 1.89 (s, 3H), 2.24 (s, 3H), 3.1-3.35 (m, 2H),4.87 (m, 1H), 6.63 (d, 1H), 7.1-7.3 (m, 4H), 7.46 (m, 2H), 7.68 (d, 2H),7.89 (d, 1H), 9.34 (s, broad, 1H). MS (EI): m/z 382(10%), 284(84%),157(8%), 155(61%), 130(26%), 129(24%). Materials: Ac-Trp-OEt Preparedaccording to procedure described under <<Peparation ofdiacetyltryptophan ethyl ester>>, Bodanszky, M and Bodanszky A, ThePractice of Peptide Synthesis (1994) p 30; Vogel's Textbook of PracticalOrganic Chemistry 5th Ed. (1989) p. 1273.

[0154] B) Preparation of Fmoc-Trp(1-Benzyl)-OH

[0155] Boc-Trp(1-Benzyl)-OH¹:

[0156] Dimethyl sulfoxide (7 ml) was added to potassium hydroxide(0.739, 13 mmol) (crushed pellets) and the mixture was stirred for 5min. Boc-Trp-OH (19, 3.3 mmol) was then added and the mixture stirredfor 1 hour. Benzyl bromide (1.13 g, 6.6 mmol) was added and the mixturecooled briefly and stirred for a further 20 hours before water (20 ml)was added. The mixture was extracted with diethyl ether (3×20 ml). ThepH of the combined aqueous phases was adjusted to 2-3 by addition of 1MHCl (20 ml) and extracted with diethyl ether (3×20 ml). Each extract waswashed with water (3×20 ml). The combined diethyl ether phases weredried with MgSO₄ and the solvent removed under reduced pressure. Theproduct was isolated as white crystalls (0.89 g, 2.3 mmol). Yield 69%.

[0157]¹H NMR (CDCl₃): δ 1.41 (s, 9H), 3.33 (m, 2H), 4.64 (m, 1H), 5.02(m, 1H), 5.24 (s, 2H), 6.95 (s, 1H), 7.01-7.38 (m, 8H), 7.59 (d, J=7.7Hz, 1H).

[0158] H-Trp(1-Benzyl)-OH:

[0159] Boc-Trp(1-Bn)-OH was dissolved in 98% TFA and stirred for 3 hoursat room temperature. Then the solvent was removed under reducedpressure. The product was isolated as an oil and used without furtherpurification.

[0160] Fmoc-Trp(1-Benzyl)-OH:

[0161] H-Trp(1-Bn)-OH (1.90 g, 6.5 mmol) was dissolved in a 10% solutinof Na₂CO₃ in water (21 ml, 20 mmol). Dioxane (15 ml) was added and themixture was stirred in an ice-water bath. 9-Fluorenylmethylchlorocarbonate (1.69 g, 6.5 mmol) was added in small portions andstirring was continued at ice-water bath temperature for 4 hours andthen at room temperature for 8 hours. The reaction mixture was pouredinto water (400 ml) and extracted with ether (3×200 ml). The combinedether phases were dried with MgSO₄ and the solvent removed under reducedpressure. The product was purified by chromatography on silica gel insolvent A (Solvent A=Ethylacetate: Methanol=4:1). After purification theproduct was obtained as a white crystalline compound. The yield was2.429 (72%).

[0162]¹H NMR (400 MHz, CDCl₃): δ 3.34 (m, 2H) 4.18 (m, 1H) 4.37 (m, 2H),4.78 (s, 1H), 5.19 (s, 2H), 5.31 (d, 1H), 6.91-7.74 (m, 19H). Materials:Boc-Trp-OH BACHEM no A-2360 Fmoc-ONSu Fluka no 46920 Trifluoroaceticacid Fluka no 91700/KEBO no 1.8341-100

[0163] Reference 1:

[0164] Heaney, H., and Ley, S. V. J. Chem. Soc. Perkin 1. (1973) 499-500

[0165] C) Preparation of Fmoc-Trp(2-Nps)-OH

[0166] To a solution of 2.0 g (4.7 mmole) Fmoc-L-tryptophan in 12 mldioxane, 0.87 g (4.6 mmole) of 2-nitrophenyl-sulfenylchloride (2-Nps-Cl)in 25 ml dioxane was added under stirring at room temperature. Afterstanding for 3 days, 50 ml ethyl ether was added to the reaction mixtureand the solvent was evaporated. The product was purified bychromatography on silica gel in solvent A (Solvent A=Chloroform:Ethanol:Neptane—1:1:1). R_(f) 0.43. After purification the product wasobtained as a yellow-brown crystalline compound. The yield was 2.5 g(89%).

[0167] HPLC (C18): t_(R) 8.3 min, 85-100% B in 20 min. (A:H₂O+0.1% TFA;B:CH₃CN+0.1% TFA).

[0168]¹H NMR (DMSO-d₆): δ 3.16 (m, 1H), 3.38 (m, 1H), 4.00-4.10 (m, 3H),4.19 (m, 1H), 6.72 (d, J=8.l Hz, 1H), 7.03 (t, J=7.3 Hz, 1H), 7.18 (t,1H), 7.22-7.49 (m, 7H), 7.60 (dd, J=7.3 and 12.1 Hz, 2H), 7.86 (m, 3H),8.24 (d, J=8.1 Hz, 1H), 11.51 (s, 1H)

[0169] After incorporation of Fmoc-Trp(2-Nps)-OH into a peptide, MSelectrospray analysis confirmed the expected molecular weight.Materials: Fmoc-Trp-OH BACHEM No B-1445/ SENN No 020192-Nitrophenylsulfenyl chloride Fluka No 73740

[0170] D) Oxidation of Fmoc-Trp(2-Nps)-OH

[0171] To a solution of 1.12 g (1.9 mmol) Fmoc-Trp(2-Nps)-OH in 15 mlglacial acetic acid, was added 12 ml 30% H₂O₂under stirring at roomtemperature. The reaction mixture was heated for 2 hours at 65° C. Theprecipitate was collected, added water and lyophilised. The yield was0.59 g (52%). The product was obtained as a yellow crystalline compound.

[0172] HPLC (C18): t_(R) 6.4 min, 85-100% B in 20 min. (A:H₂O+0.1% TFA;B:CH₃CN+0.1% TFA).

[0173]¹H NMR (DMSO-d₆): δ 3.25 (dd, J=9.0 and 14.5 Hz, 0.5H), 3.54 (m,1H), 3.77 (dd, J=5.9 and 14.3 Hz, 0.5H), 4.01-4.26 (m, 3H), 4.32 (m,0.5H), 4.40 (m, 0.5H), 7.00-7.98 (m, 15H), 8.23 (m, 1H), 8.35 (m, 1H),8.56 (d, J=8.1 Hz, 1H), 11.08 (s, 0.5H), 11.17 (s, 0.5H).

[0174] After incorporation of Fmoc-Trp(2-NpsO₂)-OH into a peptide, MSelectrospray analysis revealed that the oxidation of Fmoc-Trp(2-Nps)-OHhad been incomplete; the product was a circa 3:1 mixture of thesulfoxide Fmoc-Trp(2-NpsO)-OH and the sulfone Fmoc-Trp(2-NpsO₂)-OH.Proton NMR indicates a 1:1 mixture of the two compounds based on doublesets of signals for β-, α- and carboxyl-protons.

[0175] E) Preparation of Fmoc-Trp(2-Pmc)-OH

[0176] Method I:

[0177] By transferral of the Pmc-group from Fmoc-Arg(Pmc)-OH

[0178] Fmoc-Arg(Pmc)-OH (0.5 g, 0.75 mmol) and Fmoc-Trp-OH (0.43 g, 0.1mmol) was dissolved in 10 ml 100% TFA and heated at 30° C. for 1.5hours. After evaporation of TFA, Fmoc-Arg-OH was removed by columnchromatography on silica gel with heptane/ethyl acetate 2:1 as mobilephase. Fmoc-Trp(2Pmc)-OH was isolated by preparative HPLC (C18, 70-100%B in 15 min., t_(R) 14.8 min, (A: H₂O+0.1% TFA; B: CH₃CN +0.1% TFA)).Isolated yield 130 mg (0.19 mmol, 25%).

[0179]¹N NMR (400 MHz, CDCl₃): δ 1.30 (s, 6H), 1.79 (t, 2H), 2.07 (s,3H), 2.43 (s, 3H), 2.48 (s, 3H), 2.59 (t, 2H), 3.03 (m, 1H), 3.25 (m,1H), 4.1-4.3 (m, 3H), 4.42 (m, 1H), 6.53 (d, 1H), 7.15-7.78 (m, 12H),8.90 (s, 1H) Materials: Fmoc-Arg(Pmc)-OH BACHEM no B-1670 Fmoc-Trp-OHBACHEM no B-1445/SENN no 02019 Trifluoroacetic acid KEBO no1.8341-100/Fluka no 91700

[0180] Method II:

[0181] By transferral of the Pmc-group from phenylethylguanidyl-Pmc

[0182] 2,2,5,7,8-Pentamethylchroman:

[0183] References:

[0184] Robert Ramage, Jeremy Green and Alexander J. Blake, TerahedronVol. 47, No. 32, pp. 6353-6370, 1991. Reaction:

Chemicals: Substance Quantity MW mmoles eqv. Source2,3,5-Trimethylphenol 50.03 g 136.20 367.33 1.0 Fluka Isoprene 25.09 g68.12 368.32 1.0 Jannsen Chimica ZnCl₂ 5.94 g 136.29 43.58 0.12 FlukaAcetic acid 47 ml — — — KEBO lab

[0185] Procedure:

[0186] 2,3,5-Trimethylphenol (50.03 g, 0.367 moles), isoprene (25.09 g,0.368 moles) and fused zinc chloride (5.94 g, 0.044 moles) was stirredwith anhydrous acetic acid (47 ml) for 14 hours at room temperature. Thecloudy red coloured mixture was then gradually heated and it becameclear. Upon refluxing the reaction mixture turned black, and after 8hours of reflux it was cooled to room temperature. The reaction mixturewas poured into 250 ml water and the black oil separated. The water wasextracted with pentane (3×200 ml) and the combined organic phases washedwith Claisen's alkali (2×150 ml), water (3×250 ml) and brine (2×200 ml),dried over CaCl₂ and evaporated to a brown oil under reduced pressure.The crude product was distilled at 0.48 mBar affording the product as apale yellow liquid (36.90 g, 49% yield); b.p. 82-96° C. (0.48mBar); >95% pure (GC).

[0187] Results:

[0188] The product was isolated as a pale yellow liquid which solidifiedupon cooling in 49% yield.

[0189]¹H NMR (CDCl₃, 400 MHz): δ=1.30 (6H,s, 2×CH ₃), 1.78 (2H, t, J=7Hz, CH₂), 2.07 (3H, s, CH₃), 2.15 (3H, s, CH₃), 2.19 (3H, s, CH₃), 2.59(2H, t, J 7 Hz, CH₂), 6.54 (1H, s, aromatic H).

[0190]¹³C NMR (CDCl₃, 400 MHz): δ=11.42 (CH₃), 18.91 (CH₃) 19.84 (CH₃),20.49 (CH₂), 26.97 (2×CH₃), 32.79 (CH₂), 73.10 (C(CH₃)₂), 116.67 (Ar—C),122.03 (Ar—C), 122.29 (Ar—C), 133.44 (Ar—C _(—), 134.70 (Ar—C), 151.72(Ar—C)

[0191] MS (GC/MS):

[0192] m/z=204(100), 189(14), 149(91).

[0193] 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Chloride:

[0194] To a solution of 2,2,5,7,8-pentamethylchroman (3.39 g, 16.6 mmol)in 30 ml dichloromethane at —8° C. was added under stirringchlorosulfonic acid (3.98 g, 34.2 mmol) in 30 ml dichloromethane within3 minutes. The mixture was stirred at −8° C. for 15 minutes and at roomtemperature for 2.5 hours. The reaction mixture was carefully shakenwith 50 ml dichloromethane and 100 ml ice a couple of times and thephases separated. The crude product contained circa 16% of startingmaterial as judged by ¹H NMR. When hot pentane was added to the crudeproduct, a dark oil was formed which was removed by decanting. Theproduct was then isolated by crystallisation from pentane as a lightbrown powder (2.80 g, 9.3 mmol). Yield 56%.

[0195]¹H NMR (CDCl₃) δ 1.34 (s, 6H), 1.85 (t, J=7.0 Hz, 2H) 2.14 (s,3H), 2.60 (s, 3H), 2.62 (s, 3H), 2.68 (t, J=7.0 Hz, 2H).

[0196] 2-Phenylethylquanidine Hemisulfate:

[0197] 2-Phenylethylamine (8.49 g, 70.1 mmol) and S-methylisothioureasulfate (9.43 g, 33.9 mmol) was dissolved in 100 ml destilled water. Airwas passed over the reaction mixture and through 50% NaOH (500 ml) andthen through 5% cuprous sulfate (250 ml). The reaction mixture washeated at reflux for 5 hours. Evaporation of the solvent yielded a whitepowder. The product was isolated by crystallisation from 96% ethanol,washed with cold acetone and diethyl ether and dried in a desicator.After three crystallisations, the product contained only minor amountsof starting material. The reaction yielded 61.5% (9.14 g)2-phenylethylguanidine hemisulfate.

[0198]¹H NMR (D₂O): δ 2.87 (t, J=6.6 Hz, 2H), 3.44 (t, J=6.6 z, 2H),7.24-7.38 (m, 5H)

[0199] Phenylethylguanidyl—Pmc:

[0200] Reaction:

[0201] Ian Michael Eggleston, Ph.D. thesis, University of Oxford, 1990.Reaction:

Chemicals: Substance Quantity MW mmoles eqv. Source 2-Phenylethyl- 7.40g 212.24 34.87 1.5 B11-01 guanidine hemisulfate Pmc-SO₂—Cl 7.00 g 302.0723.17 1.0 B15-02 CH₂Cl₂ 150 ml — — — KEBO lab

[0202] Procedure:

[0203] 2-Phenylethylguanidine hemisulfate (3) (7.40 g, 34.87 mmoles) wassuspended in 6M NaOH (80 ml) and extracted into chloroform (2×80 ml).After evaporation of the solvent in vacuo the oily residue wasco-evaporated with benzene (2×10 ml). The free guanidine (3b) wasdissolved in 75 ml dichloromethane in a 250 ml round bottomed flaskequipped with a magnetic stirring bar and a 100 ml addition funnel withpressure equaliser. The funnel was charged with Pmc-SO₂-Cl (7.00 g,23.17 mmoles) dissolved in 75 ml dichloromethane. The equipment wasflushed with nitrogen, and the reaction was performed under a weaknitrogen flow. The round bottomed flask was cooled in an ice/water bath,and the Pmc-SO₂Cl solution added over a period of 20-25 minutes. Thereaction mixture was allowed to attain room temperature overnight. Thedichloromethane was evaporated in vacuo and the residue partitionedbetween water (100 ml) and ethyl acetate (120 ml). The organic layer wasthen washed with water (100 ml). Upon cooling of the ethyl acetate theproduct appeared as a white/pale yellow powder, which was filtered offand dried in vacuo.

[0204] Results:

[0205] 3.32 g of a pale yellow powder was isolated. Theyield of thereaction is 33%. Melting point: 145-147° C.

[0206]¹H NMR (CDCl₃): δ=1.30 (6H, s, 2×CH₃) 1.80 (2H, t J 7.0 Hz, CH₂),2.09 (3H, s, CH₃), 2.51 (3H, s, CH₃), 2.52 (3H, s, CH₃), 2.61 (2H, t J6.6 Hz, CH₂), 2.81 (2H, t J 7.0 Hz, CH₂), 3.43 (2H, m, CH₂), 6.21 (3H,broad s, 3×NH), 7.12-7.25 (5H, m, aromatic protons).

[0207] MS: m/z=429(6), 204 (37), 149(100), 105(24), 92 (37).

[0208] Fmoc-Trp (2-Pmc)-OH: Reaction:

Chemicals: Substance Quantity MW mmoles eqv. Source Fmoc-Trp-OH 4.14 g426.49 9.71 1.0 SENN N^(G)-(6-SO₂-Pmc)-2- 2.08 g 429.59 4.84 1.0 BEH-B17phenylethylguanidine Trifluoroacetic acid 25 ml — — — Fluka

[0209] Procedure:

[0210] N^(G)-(6-SO₂-Pmc)-2-phenylethylguanidine (2.08 g, 4.84 mmoles)and Fmoc-Trp-OH (4.149, 9.71 mmoles) was stirred with trifluoroaceticacid (25 ml) at room temperature for 2 hours. The reaction mixture wasthen evaporated in vacuo and the residue partitioned between chloroformand 1 M hydrochloric acid. By cooling of the chloroform solution theexcess of Fmoc-Trp-OH could be removed by filtration.

[0211] The product was purified by flash chromatography (ethylacetate/heptane, 1:1).

[0212] Results:

[0213] The title compound was isolated as a white powder in 26% yield.Materials: Chlorosulfonic acid Fluka no 26388 Phenylethylamine Fluka no77900 S-Methylisothiourea sulfate Fluka no 67730 Fmoc-Trp-OH BACHEM noB-1445/ SENN no 02019 Trifluoroacetic acid KEBO no 1.8341-100/

[0214] F) Preparation of Fmoc-2,5,7-tritertbutyltryptophan

[0215] 2,5,7-tritertbutyltryptophan

[0216] Tryptophan (4.00 g, 19.59 mmol), trifluoroacetic acid 98% (60 ml)and tert-butanol (15.54 g, 209.66 mmol) were mixed. The reaction mixturewas stirred at room temperature for 48 hours. The trifluoroacetic acidwas evaporated. The residue was suspended in 40 ml distilled water, andthe pH adjusted to neutral with addition of sodium hydrogen carbonate.The crude product was obtained by filtration. Crystallisation from 50%ethanol affored the product as a white powder (85-90% pure).

[0217]¹H NMR (CDCl₃): δ=1.34 (9H, s, 3 CH₃), 1.46 (9H, s, 3 CH₃), 1.49(9H, s, 3CH₃), 7.45 (1H, s, CH arom), 7.18 (1H, S, CH arom), 5.29 (1H,s, NH).

[0218] Fmoc-2,5,7-tritertbutyltryptophan:

[0219] The title compound was prepared as described forFmoc-Trp(1-benzyl)-OH.

EXAMPLE 3

[0220] Bioactivity of Lactoferricin Analogs

[0221] Synthesis of the Analogs

[0222] All the peptides were synthesized on a 9050 Millipore AutomaticPeptide Synthesizer using Fmoc protection and activation withpentafluorophenyl (Pfp)esters or in situ activation with the couplingreagent HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate). In the case of coupling with pentafluorophenylesters, 1-HOBt (1-hydroxy-benzotriazole) was added to catalyse thereaction, and when using the coupling reagent HATU the reaction was basecatalysed with DIPEA (diisopropylethylamine). All amino acids withreactive side chains were protected with acid labile protecting groupsand cleaved upon treatment with TFA (trifluoroacetic acid) containingscavengers. (See below for scavenger mixture). At the same time thepeptides were cleaved from the solid support on treatment with the TFAsolution.

[0223] A) Attachment of the First Amino Acid to the Solid Support whenSynthesizing All D-Peptides

[0224] The solid support PAC-PEG-PS (Peptide Acid—Poly EthyleneGlycol—Poly Styrene resin) (1 eq.) was mixed together with Fmoc-D-aminoacid-OPfp (5 eq.) and DMAP (dimethylaminopyridine) (1 eq.) in a smallvolume of DMF (dimethylformamide) and left to swell for 30 minutes. Thesolution was then stirred slowly for 4½ hours. Ac₂O (acetic acidanhydride) (2.5 eq.) and DMAP (0.1 eq.) were then added to the solutionin order to acetylate any remaining hydroxyl groups on the solidsupport. The solution was then stirred for another hour. The solidsupport with the C-terminai amino acid attached was isolated byfiltration and washed several times on the filter with DMF. The solidsupport was then used in the synthesis of the target peptide on the 9050Millipore Automatic Peptide Synthesizer.

[0225] B) Acetylation of the N-Terminal H₂N-Group using Acetic AcidAnhydride

[0226] The peptide-resin complex was dissolved in a small volume of DMFand treated with an excess of acetic acid anhydride (20 eq.) and DMAP (5eq.) for four hours while slowly stirring the solution with a smallmagnet. Complete acetylation was verified by a ninhydrin test/Kaiser'stest (see below).

[0227] C) Ninhydrin Test/Kaiser's Test

[0228] Less than 1 mg of the peptide-resin complex was treated withsmall equal volumes of a 5% ninhydrin solution in ethanol, a solution of80 g phenol in 20 ml ethanol and a solution of dried, distilledpyridine. The reaction mixture was heated for two minutes at 110° C.,and investigated under a microscope. (In this test a yellow reactionmixture indicates successful acetylation, while a blue solutionindicates still free amino groups.)

[0229] D) Cleavage of Acid Labile Protecting Groups

[0230] Cleavage of acid labile protection groups and cleavage of thepeptides from the solid support was achieved using a mixture of 2%anisol, 2% ethandithiol (EDT), 2% water and 2% phenol in TFA, and withcleavage times of no more than four hours. The solid support was thenremoved by filtration and the peptide precipitated in diethyl ether. Theether solution containing TFA was removed using a pasteur pipette, andthe peptide was washed several times with diethylether and dried underhigh vacuum.

[0231] E) Purification

[0232] The peptides were purified by HPLC using a C18-reversed phasecolumn (*) and a mixture of water and acetonitrile (both added 0.1% TFA)as mobile phase. Selected wavelength for detection of peptide fractionswas 254 nm.

[0233] (*) PrePak®Cartridge 25×100 mm. DeltaPak™ C18 15 μm 100 Å.(Waters Corporation.)

[0234] F) Analysis

[0235] All peptides were analysed for impurities on an analytical HPLCC18-reversed phase column using a mixture of water and acetonitrile(both added 0.1% TFA) as mobile phase. The molecular weight of thepeptides were determined by positive ion electrospray ionization massspectrometry (VG Quattro Quadrupole)

[0236] Amino Acid Derivatives used in Synthesis of Both L- and D-Analogsof Lactoferricin Fmoc-AlaPEG-PS (solid support) Fmoc-Lys(tBoc)-OPfpFmoc-Arg(Pbf)-OH Fmoc-Met-OPfp Fmoc-Arg(Pmc)-OHFmoc-β-(2-naphthyl)-alanine-OH Fmoc-Asn(Trt)-OPfp Fmoc-Phe-OPfpFmoc-Cys(Acm)-OPfp Fmoc-Ser(tBu)-OPfp Fmoc-Gln-OPfp Fmoc-Thr(tBu)-OPfpFmo-Glu(OtBu)-OPfp Fmoc-Trp-OPfp Fmoc-Gly-OPfpFmoc-Tyr(tBu)-OPfpFmoc-Leu-OPfp

[0237] Amino acid derivatives were purchased from either Bachem,MilliGen/Biosearch (Division of Millipore) or PerSeptive Biosystems.

[0238] Antimicrobial Activity of Alanine Scan Peptides Containing aTryptophan-Pmc Residue

[0239] During deprotection of acid labile protecting groups and cleavageof the peptide from the resin with trifluoro acetic acid, a sidereaction involving transfer of the Pmc(2,2,5,7,8-pentamethylchroman-6-sulphonyl group) protecting group fromarginine to the second position of the indole of tryptophan wasobserved. Isolation of these byproducts have been done, and the resultsfrom MIC analyses are given in Table 2. This table also shows theresults of an alanine scan performed on LFB with no Pmc groups.

[0240] During an alanine scan, a series of peptides are produced whereinsuccessive amino acids have been substituted by alanine.

[0241] The sequence of native bovine lactofericin from amino acids 17 to31 (LFB 17-31) isH₂N-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-COOH(SEQ ID No. 1). TABLE 2 MIC results for alanine scan peptides with a Pmcgroup attached to one of the two tryptophan residues. Results also shownfor an alanine scan performed on LFB with no Pmc groups. Pmc Without PmcMIC MIC MIC MIC Peptide E. coli S. aureus E. coli S. aureus LFBA 1 8.7510   70 >200 LFBA 2 11.25 10   80 >200 LFBA 3 7.5  7.5 25 100 LFBA 4 1527.5 70 >200 LFBA 5 10  50(*) 120 >200 LFBA 6 25 17.5 >200 >200 LFBA 720  7.5 30 150 LFBA 8 15 17.5 >200 >200 LFBA 9 10 12.5 55 >200 LFBA 1020 22.5 140 >200 LFBA 11 22.5 22.5 70 >200 LFBA 12 20 20   50 >200 LFBA13 15 15   50 >200 LFBA 14 15 17.5 25 160 LFBA 17-31 10 10   50 100

[0242] The results show that a Pmc group attached to one of thetryptophan residues increases the activity four times for E. coli. Evenmore marked is the effect on S. aureus. This gram positive bacteria wasfound to be nearly totally resistant to all the alanine scan analogs,but shows now a MIC between 10 and 22.5 μg/ml. This represents a tenfold increase in antibacterial activity relative to native LEB 17-31.The tryptophan-Pmc residue, which is hydrophobic, seems therefore toincrease the peptide; affinity for the hydrophobic parts of thebacterial cell membrane to such an extent that the antibacterialactivity of the peptide is no longer so sequence dependent as for thepeptide without this residue.

[0243] Comparison of Antimicrobial Activity Between Native BovineLactoferricin (LFB 17-31) and Enantio-, Retro- and Retro-Enantio LFB17-31 and these Same Peptides Incorporating a Tryptophan-Pmc Residue

[0244] Peptides containing a Pmc-group transferred from an arginineresidue to a tryptophan residue was also isolated after synthesis ofEnantio-, Retro- and Retro-Enatio LFB 17-31. TABLE 3 MIC results fornative bovine lactoferricin (LFB 17-31), Enantio-, Retro- andRetro-Enantio LFB 17-31 and for these peptides with a Pmc group attachedto one of the two tryptophan residues. MIC % Hemolysis Peptide MIC E.coli S. aureus 10 μg/ml Native LFB 17-31 50 100 2.6 Enantio LFB 17-31  7.5 60 3.05 Retro LFB 17-31 80 200 2.01 Retro-Enantio LFB 17-31   6.25 80 3.31 LFB 17-31 Pmc 10 10 2.8 Enantio LFB 17-31 Pmc    7.5(*)100 3.17 Retro LFB 17-31 Pmc 10 10 2.5 Retro-Enantio LFB 17-31   7.512.5 5.28 Pmc

[0245] The Enantio peptide, which is the exact mirror image of thenative peptide, shows remarkable improvements in antibacterial activity.(In fact, this peptide shows the same activity as the native peptide LFB17-31 with a tryptophan-Pmc residue, in the case of E. coli.)Configurationally this means that an all-D-amino acid analog of LFB17-31 interacts better with the chiral phospholipids of the bacterialcell membrane than the native all-L-amino acid peptide LFB 17-31. It mayalso imply that this Enantio peptide is more resistant to degradableproteases of the bacteria.

[0246] The Retro peptide, with an inverted sequence in respect to LFB17-31, shows no improvements in antibacterial activity, which isconsistent with the theory of the antibacterial activity of LFB 17-31being sequence specific. This peptide is not really an isomer of bovinelactoferricin since the amino acid sequence is totally different. Thelow antibacterial activity of this peptide does therefor not come as anysurprise.

[0247] A remarkably high antibacterial activity against E. coli wasobserved for the Retro-Enantio peptide which, as already mentioned,adopts the same a-helical conformation as the native peptide LFB 17-31,except that the amide bonds point in opposite directions. Theall-L-amino acid stereoisomer, Retro LFB 17-31, shows low antibacterialactivity. The reason may be that all-D-amino acid peptides eitherinteract more strongly with the chiral phospholipids of the bacterialcell membrane or that they are more resistant to proteases than theirall-L-amino acid counterparts.

[0248] The activity of the peptides against S. aureus is not as high asobserved for E. coli, indicating that the interactions of the peptideswith the lipopolysaccharide layer of gram negative bacteria might bestronger than the interactions with the lipid cell membrane of grampositive bacteria.

[0249] The activity of the tryptophan-Pmc containing peptides do notshow the same differences between all-D- and all-L-amino acid isomers aswas observed for the peptides without the Pmc group. The effect of thetryptophan-Pmc residue seems to be more pronounced than theconfigurational effects found among the peptides without this residue,especially in the case of S. aureus. Most noticable is the tremendousincrease in activity of the Retro-Pmc peptide. The activity of thispeptide is increased eight times in the case of E. coli and more thanten times in the case of S. aureus just because of the tryptophan-Pmcresidue.

[0250] The improvements observed upon Pmc modification in the case of E.coli is neglible, but the modification increases the activity against S.aureus about six times. The gram positive bacteria are obviously morevulnerable towards tryptophan-Pmc containing peptides than their nontryptophan-Pmc containing counterparts.

[0251] Antimicrobial Activity of Tryptophan Modified Human (LFH),Porcine (LFP) and Caprine (LFG) Lactoferricin

[0252] The results from the alanine scan of bovine lactoferricin (LFB17-31) showed that the two tryptophan residues in positions six andeight could not be substituted by alanine without a major loss ofantibacterial activity. Examination of the similar sequence parts ofnative LFH, LFP and LFG lactoferricin shows that these peptides lack thetryptophan residue in position eight, but have during evolutionconserved the tryptophan residue in position six. We have synthesizedLFH, LFP and LFG analogs with a tryptophan residue substituted in theposition eight to see if the antimicrobial activity of these peptidescould be increased. The MIC values for the native sequences are given inTable 4, together with the tryptophan modified peptides.

[0253]H₂N-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Trp-Asn-Met-Arg-Lys-Val-Arg-Gly-COOH

[0254] Sequence of modified human lactoferricin (LFHW8) Substitutedtryptophan is high-lighted. (Arg-Trp)

[0255]H₂N-Ser-Lys-Cys-Tyr-Gln-Trp-Gln-Trp-Arg-Met-Arg-Lys-Leu-Gly-Ala-COOH

[0256] Sequence of modified caprine lactoferricin (LFGW8). Substitutedtryptophan is high-lighted. (Arg-Trp)

[0257]H₂N-Glu-Lys-Cys-Leu-Arg-Trp-Gln-Trp-Glu-Met-Arg-Lys-Val-Gly-Gly-COOHTABLE 4 MIC results for tryptophan modified human (LFHW8) and caprine(LFGW8) lactoferricin. (MIC values for native LFB 17-31 and nativesequences of LFH and LFG are also listed for the sake of comparison.) %Hemolysis Peptide MIC E. coli MIC S. aureus 10 μg/ml LFHW8 110 >1000 2.5LFPW8 500 >1000 2.9 LFGW8 Y13 110 >1000 NT LFGW8 500 >1000 2.7 NativeLFB 17-31  50    100 2.6 Native LFH >1000    >1000 NT NativeLFP >1000    >1000 Native LFG 750 N.T 2.4

[0258] Both LFHW8 and LFGW8 show improvements in activity against E.coli compared to the native sequences of the same peptides.

[0259] Antimicrobial Activity of LFH, LFP and LFG with a Tryptophan-PmcResidue

[0260] During acidic cleavage of the peptide (either with or without theabove modifications to the native sequence) from the resin and cleavageof acid labile protecting groups, a byproduct with a Pmc-group attachedto one of the tryptophan residues was isolated and analysed forantibacterial activity. The results are shown in Table 5. TABLE 5 MICresults for LFH, LFG and LFP with a Pmc group attached to one of the twotryptophan residues, for LFC Pmc and LFH Pmc, the PMC group will beattached to the only available tryptophan. Peptide MIC E. coli MIC S.aureus LFG Pmc 25 25 LFH Pmc 25 50 LFHW8 Pmc 25 20 LFGW8 Pmc 50 75 LFPW8Pmc 20 50 LFHW8 Y13Pmc 25 20

[0261] As for all tryptophan-Pmc containing peptides analysed so far,these peptides generally show remarkable improvements in antibacterialactivity against both E. coli and S. aureus.

[0262] Antimicrobial Activity of Tryptophan Rich Analogs of BovineLactoferricin (LFB 17-31)

[0263] The alanine scan showed that the two tryptophan residues in thesequence of bovine lactoferricin 17-31 were absolutely essential to theantibacterial activity of the peptide. Alanine substitution of any ofthese two residues led to a major loss of antibacterial activity. Thealanine scan also showed that the nonessential amino acids in thesequence of bovine lactoferricin 17-31 were the three residues Cys(3),Gln(7) and Gly(14). Based on this knowledge we therefore synthesized aseries of five tryptophan rich analogs of bovine lactoferricin 17-31with one, two or three of the nonessential amino acids substituted bytryptophan. This technique of performing an alanine scan and thenreplacing seeming non-essential amino acids with Tryptophan or otherbulky and/or lipophilic amino acids can be used to enhance thecytotoxicity of peptides generally, and is not limited to lactoferricin.The sequences of the tryptophan rich bovine lactoferricin analogs areshown below. LFBW3:H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-COOHLFBW14:H₂N-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOHLFBW3, 14:H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOHLFBW3, 7, 14:H₂N-Phe-Lys-Trp-Arg-Arg-Trp-Trp-Trp-Arg-Met-Lys-Lys-Leu-Trp-Ala-COOHLFBW4, 10:H₂N-Phe-Lys-Cys-Trp-Arg-Trp-Gln-Trp-Arg-Trp-Lys-Lys-Leu-Gly-Ala-COOH

[0264] TABLE 6 MIC results for five tryptophan rich analogs of bovinelactoferricin (LFB 17-31) together with native LFB 17-31. Peptides MICE. coli MIC S. aureus LFB 17-31 50 100 LFBW3 20 20 LFBW14 20 25 LFBW3,14 10 10 LFBW3, 7, 14 20 20 LFBW4, 10 5 10

[0265] Substitution of nonessential amino acids in the sequence of LFB17-31 by tryptophan residues improves the antibacterial activity ofthese peptides by at least two times that of the native sequence in thecase of E. coli and by four times in the case of S. aureus.

[0266] Peptide W3714, with three additional tryptophan residues (a totalof five tryptophan residues in the peptide), has decreased activity.This is probably more a result of a solubility problem, this peptidebeing less soluble in aquous solutions and therefore giving lowerconcentrations than calculated. This has been physically observed duringMIC testing procedures when the peptide tended to precipitate at highconcentrations.

EXAMPLE 4

[0267] Antitumoral Effects of Different Peptides

[0268] Cyclic LFB 17-41 was from Morinaga, Milk Industri, JapanCytotoxicity

[0269] Different murine and human tumor cells (4×10⁶) were applied in96-well culture plates (Costar) in a volume of 0.1 ml RPMI 1640 medium.Peptide solutions (0.1 ml) were added and the plates incubated at 37° C.for 30 minutes, 4 hours or 24 hours. The cytotoxicity was measured usingthe MTT method (Mosmann et al., J. Immunol. (1986) 136, 2348-2357).

[0270] Electron Microscopy

[0271] Scanning Electron Microscopy (SEM)

[0272] For scanning electron microscopy, Meth A cells were cultivated ina 12 well culture plate and treated with different peptides as describedabove. Cells were fixed in McDowell's fixative postfixated in 1% OsO₄,dehydrated and critical point dried according to standard procedures.The cells were examined in a Jeol JSM-5300 Scanning microscope.

[0273] Transmission Electron Microscopy (TEM)

[0274] Meth A cells were harvested from 12 culture plates by aspirationand fixed in McDowell's fixative overnight, followed by postfixation,dehydration and embedding in Epon Araldite according to standardprocedures. Ultrathin sections were cut on a Reichert Ultracut S andthen contrasted in 5% Uranyl acetate and Reynold's lead citrate.Sections were examined in a Jeol LEM-1010 transmission electronmicroscope.

[0275] Experimental Animals

[0276] Specific pathogen-free CB6F1 (Balb/c×C57 BL/6) female mice ofabout 8 weeks of age were obtained from Charles River (Germany). Themice were kept on standard laboratory chow and water. Tumor bearing micewere serologically screened for viral (LDH, CMV) and mycoplasmicinfection and in all cases tested negative.

[0277] Tumors

[0278] Meth A is a non-adhesive murine sarcoma cell line [Sveinbjørnssonet al, (1996) BBRC 223: 643-649] syngenic in Balb/c and was maintainedin vitro in RPMI 1640 containing 2% Foetal calf serum. Cells in thegrowth phase were harvested and washed in fresh medium and injectedsubcutaneously into the abdominal region of the mice. Each mousereceived a single inoculation of 5×10⁶ viable tumor cells in RPMI 1640.

[0279] Results

[0280] In vitro

[0281] Cytotoxicity

[0282] Lactoferricin B Derivatives

[0283] A) Meth A

[0284] 1. Cyclic and Linear LFB

[0285] The cytotoxic effect of cyclic and linear LFB (17-41) on Meth Acells was studied. Linear LFB, with the cysteins protected with Acm,killed the Meth A cells (1×10⁴/ml) effectively at concentrations higherthan 0.6 mg/ml after 4 h incubation (FIG. 2). Cyclic LFB, which is anenzymatically cleaved fragment of bovine lactoferrin effectively killedmore than 99% of the cells at concentrations higher than 0.8 mg/ml.

[0286] 2. LFB derivatives

[0287] LFB derivatives with different lengths and modifications weretested for their cytotoxic properties. Meth A cells were incubated withdifferent concentrations of the different LFB derivatives, for ½ hourand 4 hours. As shown in FIG. 3, Unmodified LFB 17-31 had no significantcytotoxic effect on the Meth A cells at concentrations up to 1 mg/mlafter ½ hour incubation. In this experiment it had a weak effect at 1mg/ml after 4 hours incubation (FIG. 4). The PMC modified LFB 17-31analoge killed the tumor cells at concentrations higher than 500 μg/mlafter ½ hour incubation. The same concentration was needed to achieveeffective killing after 4 hours. Linear LFB (17-41) modified with Pmcwas slightly more effective than Pmc modified LFB 17-31.

[0288] In the figures “−” denotes no Pmc and “+” denots with PMCmodification.

[0289] A shorter sequence LFB 20-29 modified with PMC killed more than90% of the cells at 250 μg/ml. An LFB 17-31 analogue (alaninesubstitution in position 8) that was modified with PMC and N-terminalFmoc protected was effective at concentrations higher than 100 μg/mlafter ½ hour and at 50 μg/ml after 4 hours. An Fmoc protected LFBpeptide (Alanine substitution in position 8) killed most of the cells at250 μg/ml at ½ hour and 4 hours (FIGS. 5 and 6). So it seems that acombination of Fmoc and Pmc modification enhanced the cytotoxic effectof LFB more than each of the two modifications alone. The retro LFBanalog was also tested. The retro-Pmc-modified LFB 17-31 also possessedan enhanced cytotoxic effect compared to unmodified LFB 17-31 (FIGS. 5and 6).

[0290] B) Human Promelocytic Leukemia Cell Line HL60.

[0291] The cytotoxic effect of LFB 17-41 (PB), LFB 14-31 (P1), LFB 14-31Pmc (P2), LFB 17-31 (P3) and LFB 17-31 Pmc (P4) on human HL 60 cells wasstudied. LFB 14-31 and LFB 17-31 showed no cytotoxic effect at theconcentration tested whereas LFB 17-41 possessed a weak concentrationdependant cytotoxic effect. The LFB 17-31 Pmc peptide induced a markedlystronger effect (appr. 5 fold higher) than the other peptides tested.See FIG. 7.

[0292] 3. EM Studies

[0293] The SEM and TEM results show that the cell membranes are stronglydisrupted by lactoferricin peptides, resulting in effective release ofintracellular material. The lysis seems to be very rapid, i.e. withinminutes by the most effective peptides.

[0294] In Vivo

[0295] 1. Tumor Regression

[0296] Murine Meth A Fibrosarcoma

[0297] After a single inoculation of 5×10⁷ viable Meth A cells,different LF peptides were injected intratumorally (LFB-14-31, LFB 17-31Pmc, 500 μg in a 50 μl dose; LFB 17-31, 1000 μg in a 50 μl dose), on day7 and day 10. LFB 14-31 was also injected intraperitoneally (PBI), 500μg/ml. Saline only was injected in the control mice (50 μl) (K1,K2,K3).The tumor diameter (mean of transversal and longitudinal) were measuredwith an electronic calipper.

[0298] The in vivo Effect of LFB 17-31. LFB 17-31pmc and LFB on MurineMethA Fibrosarcoma

[0299] As shown in FIG. 8, all three peptides tested, LFB 17-31 (PB),LFB 14-31 pmc (P2), LFB 14-31 (Pi), induced regression of the Meth Atumors, after treatment on day 7 and 10. “Diam. mm.” refers to diameterof the tumours.

[0300] Interestingly, tumors were also eradicated in the mice that weretreated intraperitoneally with LFB 14-31 (PBI). Mice treated with salineonly are represented as K1, K2 and K3.

EXAMPLE 2 Murine Melanoma B16F10

[0301] After a single inoculation of 5×10⁶ viable B16F10 murine melanomacells, D-LFB A7 Pmc-NH₂ was injected intra-tumorally in the tumors onday 10 and 12 (500 μg/injection in 50 μl saline). Saline only wasinjected in the control mice (50 μl). The tumor diameter (mean oftransversal and longitudinal) were measured every second day with anelectronic calipper.

[0302] The in vivo Effect of D-LFB A7 Pmc-NH2 on Murine Melanoma B16F10

[0303] As shown in FIG. 9, D-LFB A7 Pmc-NH₂ (pep) was able toeffectively induce regression of the solid tumors. The y axis representsthe diameter of the tumour in mm. Three out of five were totallyeradicated after only two injections. After six days after the firsttreatment, one of the tumor started to grow again, and 10 days after thefirst treatment a second tumor started to grow.

[0304] 2. Adaptive Immunity

[0305] After successful treatment of established Meth A tumors, somemice were kept for one month before reinoculation of tumor cells asdescribed above. In some of these mice a third inoculation of tumorcells were performed one month later than the second inoculation. Notumors were established in these mice and the mice were kept for alonger period without any effect on the normal condition of these mice.

EXAMPLE 5

[0306] The effect of chemical modification of a further moderatelyactive peptide has also been investigated. The stating peptide is afragment of bovine lactoferrin, which corresponds to residues 14-31 ofthe native sequence (see Table 1 in FIG. 1 for the full sequence). Theantimicrobial activity in the form of MIC values against E. coli and S.aureus, the toxicity expressed as the concentration which caused 50%hemolysis (EC 50) and the anti-tumour activity in the form of the numberof μg/ml of peptide required to kill 50% of MethA cells for the peptidesare shown below in Table 7. TABLE 7 MIC MIC MethA E. coli S. aureus EC50 IC 50 Peptide μg/ml μg/ml (μM) μM/ml LFB 14-31 70 >250 >404 noactivity LFB 14-31 PMC 15 20 244 14.6 LFB 14-31 A2, 6, 10, 17 202.5 >440 165 LFB 14-31 A2, 6, 10, 17 20 2.5 >165 12.8 PMC LFB 14-31 A2,6, 10, 17R4 30 20 >438 75.8 LFB 14-31 A2, 6, 10, 17R4 10 2.5 290 6.9 PMCLFB 14-31 A2, 6, 10, 17R4, >444 75.5 11 LFB 14-31 A2, 6, 10, 17R4, 3275.2 11 PMC LFB 14-31 A2, 6, 10, 10 2.5 >440 30.2 17F7R4 LFB 14-31 A2, 6,10, 10 2.5 20 7.7 17F7R4 PMC LFB 14-31 A2, 6, 10, 10 10 >440 28.117F7K16L14 LFB 14-31 A2, 6, 10, 10 2.5 89 5.2 17F7K16L14 PMC

[0307] As before, the presence of the bulky/lipophilic group PMC on oneor more of the tryptophan residues enhances the antimicrobial andanti-tumour activity. Interestingly, the presence of this artificialbulky and lipophilic group is able to selectively enhance bacteriocidalactivity, activity against S. aureus generally being more enhanced thanagainst E. coli.

EXAMPLE 6

[0308] Table 8 shows anti-bacterial activity and toxicity data for LFBbased peptides incorporating a non-genetic bulky and lipophilic amino inplace of one of the amino acids in the native sequence. Further peptidesalso incorporate a group (PMC) which increases the bulk andlipophilicity of one of the naturally occurring tryptophan residues.TABLE 8 MIC MIC % % E. coli S. aureus Hemolysis Hemolysis Peptide μg/mlμg/ml 10 μg/ml 100 μg/ml LFB 50 100 2.6 3.47 LFB Bip3 10 10 LFB Bip6 2525 LFB Bip8 15 15 LFB Bip6, 8 10 5 LFB Bip3 PMC 37.5 2.5 LFB Bip8 PMC 255 LFB Tbt3 25 5 LFB Tbt3 PMC 37.5 10 LFB Tbt6 12.5 10 LFB Tbt6 PMC 37.510 LFB Tbt8 12.5 5 LFB Tbt8 PMC 25 5 LFB Tbt6, 8 25 5 LFB Nal6 20 75 2.24.4 LFB Nal6 PMC 25 20 2.8 17.8 LFB Nal6, 8 10 20 2.8 4.9 LFB Nal8 10 503 4.7 LFB Nal8 PMC 20 10 6.96 18.86 LFB NPS-O6 20 100 2.8 4.1 LFB NPS623 50 4.2 5.9

[0309] All peptides are LFB 17-31 and modifications thereof.

EXAMPLE 7

[0310] Experiments were performed to investigate the effect of PMC andvarying peptide length on anti-tumour activity and toxicity (hemolyticactivity) The results of these experiments are presented in Table 9below. TABLE 9 Meth A IC₅₀ (μM) RBC EC₅₀ (μM) Selectivity Peptide −PMC+PMC −PMC +PMC +PMC LFB 14-31 A_(2, 6, 10, 17) 165 15 >440 118 8 LFB14-30 A_(2, 6, 10, 17) >227 14 >454 184 13 LFB 14-29 A_(2, 6, 10) >23518 >469 367 20 LFB 14-28 A_(2, 6, 10) >248 12 >438 >438 >36

[0311] The presence of a PMC group on one or more of the tryptophanresidues of an LFB peptide significantly increased its anti-tumouractivity and to a lesser extent its hemolytic activity. Surprisingly itwas found that by reducing the length of the peptide the selectivity,i.e. the anti-tumour verus the hemolytic activity of the peptideincreased.

EXAMPLE 8

[0312] Methods for the Preparation of Peptide Esters

[0313] Transesterification from Resin

[0314] Fully protected peptide esters can be obtained by base catalysedtransesterification from SASRINTM and Merrifield-like resins. Goodyields have been obtained with methanol and benzyl alcohol. The bestresults were obtained employing either KCN2, or LiBr/DBU as catalyst.

[0315] Standard Procedure for KCN-Catalysed Transesterification:

[0316] The peptide resin and the solvent employed have to be driedcarefully before use, all have to withstand prolonged KCN-treatment.Transesterification will occur, even if the solubility of KCN is low;residual salt did not disturb. The peptide resin is suspended in amixture of the desired alcohol and the cosolvent, e.g.dimethylacetamide, (usually 1:1, 10 ml/g resin). After 30 min sufficientsolid KCN is added, so that a 0.08 M solution is obtained (or at leastsaturation). After stirring for 24 hours, the resin is filtered off andwashed with the cosolvent. The catalyst must be destroyed immediately,e.g. by rigourously shaking the filtrate with sufficient solid anhydrousFeCl₂. Iron blue will flock out, it is left to settle for approx. 30 minand filtered off. The filtrate may remaine greenish. Further work-updepends on the solubility of the product, but it should be treated withwater: After removing alcohol and cosolvent, the residue is taken up inan organic solvent, e.g. ethyl acetate or chloroform, for furtheraqueous extraction to remove salts.

[0317] Direct Benzyl Esterification Of N-Acylpeptides

[0318] (p-Hydroxyphenyl)benzylmethylsulfonium derivatives (HOBMX) easilygenerate benzyl cations, which convertes N-terminal- and sidechain-protected peptides into thier benzyl esters without racemization.

[0319] General Procedure:

[0320] The petide and potassium carbonate are dissolved indichloromethane, and the mixture is stirred at room temperature. After10 min, HOBMCl is added to the solution and it is stirred for 8 hours.Inorganic salts in the reaction mixure are filtered off and the filtrateevaporated in vacuo. The residue is dissolved in toluene and washed with0.5 M NaOH aqueous solution and then with water. The organic layer isdried over anhydrous sodium sulfate and the filtrate evaporated invacuo.

EXAMPLE 9

[0321] A series of further modified peptides were prepared based onmurine lactoferrin. In the following data (see Table 10), LFM refers toresidues 17-31 of murine lactoferrin. Shorter peptides are indicated bythe notation wherein e.g. LFM 17-24 represents an 8-mer peptidecorresponding to the amino acids at positions 17 through to 24 of murinelactoferrin.

[0322] The murine equivalent of LFB is generally much less active thanits bovine equivalent, however, by modifying the peptide in accordancewith the present invention peptides with greatly enhanced anti-bacterialactivity can be prepared. LFM does not possess a tryptophan residue atposition 8, unlike its more active bovine counterpart. The inventorshave identified this residue as important to the activity of LFB andthus this substitution of asparagine for tryptophan has been made.

[0323] This substitution alone did not significantly enhance theactivity against the bacterial strains tested. Activity could be furtherenhanced by substituting one or both of the anionic residues atpositions 1 and 9 with unchanged alanine or more preferably a cationicresidue such as arginine.

[0324] By incorporating further bulky/lipophilic residues, e.g. atyrosine residue at position 13 in place of the less bulky valine and/orby modifying the tryptophan residue by incorporation of the more bulkyPMC group, peptides with good antimicrobial activity could be made.

[0325] In addition it was surprisingly found that shorter peptides basedon fragments of LFM when modified to introduce additionalbulky/lipophilic amino acids e.g. tryptophan or tyrosine and to increasethe overall charge of the peptide by replacing native residues withcationic residues such as arginine were particularly effective. TABLE 10MIC MIC % % E. coli S. aureus Hemolysis Hemolysis Peptide μg/ml μg/ml 10μg/ml 100 μg/ml LFM >1000 >1000 2.3 3.1 LFM W8 >1000 1000 2.6 4.8 LFM W8Y13 >1000 >1000 LFM A1 W8 750 >1000 2.4 3 LFM A1 W8 Y13 500 >1000 LFM A9W8 >1000 >1000 2.5 3.5 LFM A9 W8 Y13 >1000 >1000 LFM A1, 9 W8 200 >10002.8 3.7 LFM A1, 9 W8 Y13 150 >1000 LFM R1, W8 75 500 2.8 3.48 LFM R1 W8PMC >200 >200 LFM R1 W8 Y13 50 50 LFM R9 W8 500 >1000 3.1 4.59 LFM R9 W8PMC 20 50 LFM R9 W8 Y13 150 1000 LFM R1, 9 W8 25 75 3.69 LFM R1, 9 W8PMC 10 5 4.9 LFM R1, 9 W8 Y13 25 50 LFM A1 R9 W8 Y13 50 200 LFM 17-24R1, 2, 8 W3, 7Y4NH2 5 2.5 LFM 17-24 R1, 2, 8 W3, 7Y4NH2 PMC 25   1-2.5LFM 18-24 R1, 7 W2, 3, 6Y5NH2 10 0.5-1   LFM 17-25 A4R2, 8, 9W3, 7Y1NH210 5 LFM 17-25 A4R2, 8, 9W3, 7Y1NH2 PMC 20 2.5 LFM 17-26 A7R2, 8, 9W3,4, 10Y1NH2 10 2.5

EXAMPLE 10

[0326] Table 11 below illustrates the effect of further chemicalmodifications which provide peptides in accordance with the invention.TABLE 11 MIC MIC % % MethA E. coli S. aureus Hemolysis Hemolysis IC₅₀Peptide μg/ml μg/ml 10 μg/ml 100 μg/ml μg/ml LFB 50 100 2.6 3.47 500 LFBPMC 10 10 2.8 4.4 120 LFB PMC6 10 10 3.4 6 148 LFB PMC8 18 10 3.6 9.79150 LFB 18-31 80 200 LFB 18-31 PMC 10 10 LFB 19-31 200 >250 LFB 19-31PMC 10 15 LFB 20-28 A4 >100 >100 0 1.68 500 LFB 20-28 A4 120 FMOC LFB20-28 A4 35 FMOC PMC LFB 20-28 A4 15 3.9 12.6 110 PMC LFB 20-29 60 >1001.75 2.74 500 LFB 20-29 5 10 10.3 28.2 140 FMOC LFB 20-29 22.5 60.2 50FMOC PMC LFB 20-29 PMC 10 10 5.6 18.9 160 LFB 20-30 40 >100 2.16 3.1 LFB20-30 PMC 15 10 5.54 15.8 LFB 20-31 100 200 LFB 20-31 PMC 10 10 LFB A170 >200 LFB A1 PMC 8.75 10 LFB A2 80 >200 LFB A2 PMC 11.25 10 LFB A3 25100 500 LFB A3 PMC 7.5 7.5 2 3.67 130 LFB A4 7.0 >200 LFB A4 PMC 15 27.5LFB A5 120 >200 LFB A5 PMC 10 50 LFB A6 >200 >200 2.78 3.27 LFB A6 PMC25 17.5 LFB A7 30 150 500 LFB A7 PMC 20 7.5 2.1 3.8 88 LFB A8 >200 >2002.8 3.45 500 LFB A8 FMOC 60 10 2.87 7.79 LFB A8 FMOC 6.75 45.4 PMC LFBA8 PMC 15 17.5 275 LFB A9 55 >200 LFB A9 PMC 10 12.5 LFB A10 140 >200LFB A10 PMC 20 22.5 LFB A11 70 >200 LFB A11 PMC 22.5 22.5 LFB A1250 >200 LFB A12 PMC 20 20 LFB A13 50 >200 LFB A13 PMC 15 15 LFB A14 25160 500 LFB A14 PMC 15 17.5 100

EXAMPLE 11

[0327] Table 12 below illustrates the antibacterial activity as well astoxicity (% hemolysis) data and some anti-tumoural activity for furtherpeptides according to the invention. LFB stands for LFB 17-31. TABLE 12MIC MIC % % Meth E. coli S. aureus Hemolysis Hemolysis A IC₅₀ Peptideμg/ml μg/ml 10 μg/ml 100 μg/ml μg/ml LFB F4 20 200 2.4 3.2 LFB F4 PMC 2020 LFB F4K1 20 200 LFB F4K1 PMC 10 10 LFB K1 60 100 LFB K1 PMC 10 10 LFBW3 20 20 2.3 3.8 LFB W3 PMC >50 10 3.55 17.35 LFB W3, 14 10 10 3.1 5.1LFB W3, 14 20 20 PMC LFB W3, 7, 14 20 20 4.02 66.1 LFB W3, 7, 14 30 2018.1 85.9 PMC LFB W4, 10 5 10 4.45 27.8 500 LFB W4, 10 20 20 2.27 14.2110 PMC LFB W14 20 25 3 4.1 LFB W14 PMC 25 10

EXAMPLE 12

[0328] Antibacterial peptides which are active against bacterial strainswhich have been shown to demonstrate resistance to other antibiotics arepotentially very useful peptides. Table 13 below gives the antibacterialactivity and toxicity data for some preferred peptides of the invention.MRSA is methicillin resistant S. aureus and MRSE is methicillinresistant S. epidermidis.

[0329] In Table 13, LFB=LFB 17-31 unless otherwise indicated. Thepreviously identified one and three letter codes are used and inaddition, the following N-terminal modifying groups are represented:

[0330] Bz=benzyl, CHx=cyclohexyl, Ad=adamantyl TABLE 13 MIC E. coli MICS. aureus MIC MRSA MIC MRSE EC 50 Peptide μg/ml μM μg/ml μM μg/ml μMμg/ml μM μg/ml μM Bz LFB Chx LFB >20 >9.2 2.5-5   1.1-2.3 Ad LFB 7.5-10 3.3-4.5 2.5-5   1.1-2.2 LFB PMC 6 10.0 4.3 2.5 1.1 2.5 1.1 >1000 >429LFB A3 PMC 7.5 3.4 5.0 2.2 >1000 >449 LFB A7 PMC 20.0 8.8 15.5-20  6.8-8.8 ≧20 ≧8.8 17.5 7.7 >1000 LFB A3, 7 >20 >10.5 17.5-20    9.2-10.55.0 2.6 >1000 >525 LFB A3, 7 PMC 2.5 1.2 2.5 1.2 2.5 1.2 >1000 >461 LFBW3, 14 10.0 4.5 2.5 1.1 2.5 1.1 2.5 1.1 >1000 >453 LFB retro PMC 10.04.3   5-7.5 2.1-3.2 >1000 >429 LFB enantio PMC 7.5 3.2 2.5-5   1.1-2.12.5-5   1.1-2.1 2.5 1.1 >1000 >429 LFB 20-30 PMC 15.0 8.3 10.0 5.5 LFB17-27 A3, 7 R2, 11 W4, 10 10.0 5.9 2.5 1.5 0.5-1   0.3-0.6 2.5 1.5  700± 300  400 ± 200 Y1 NH2 LFB 17-27 A7 M3 R2, 11 W4, 10 10.0 5.7 0.5-1  0.3-0.6  510 ± 160 291 ± 91 Y1 NH2 LFB 17-27 A3, 7 R2, 11 W4, 10   1-2.50.5-1.3 43 ± 8 22 ± 4 Y1 NH2 PMC Bz LFB Chx LFB >20 >9.2 2.5-5   1.1-2.3Ad LFB 7.5-10  3.3-4.5 2.5-5   1.1-2.2 LFB PMC 6 10.0 4.3 2.5 1.1 2.51.1 >1000 >429 LFB A3 PMC 7.5 3.4 5.0 2.2 >1000 >449 LFB A7 PMC 20.0 8.815.5-20   6.8-8.8 ≧20 ≧8.8 17.5 7.7 >1000 LFB A3, 7 >20 >10.5 17.5-20   9.2-10.5 5.0 2.6 >1000 >525 LFB A3, 7 PMC 2.5 1.2 2.5 1.2 2.51.2 >1000 >461 LFB W3, 14 10.0 4.5 2.5 1.1 2.5 1.1 2.5 1.1 >1000 >453LFB retro PMC 10.0 4.3   5-7.5 2.1-3.2 >1000 >429 LFB enantio PMC 7.53.2 2.5-5   1.1-2.1 2.5-5   1.1-2.1 2.5 1.1 >1000 >429 LFB 20-30 PMC15.0 8.3 10.0 5.5 LFB 17-27 A3, 7 R2, 11 W4, 10 10.0 5.9 2.5 1.5 0.5-1  0.3-0.6 2.5 1.5  700 ± 300  400 ± 200 Y1 NH2 LFB 17-27 A7 M3 R2, 11 W4,10 10.0 5.7 0.5-1   0.3-0.6  510 ± 160 291 ± 91 Y1 NH2 LFB 17-27 A3, 7R2, 11 W4, 10   1-2.5 0.5-1.3 43 ± 8 22 ± 4 Y1 NH2 PMC LFM 17-26 A7R2,8, 9W3, 4, 10.0 6.2 1.0 0.6 2.5 1.5 880 ± 90 543 ± 56 10Y1NH2 LFM 17-26A7R2, 8, 9W3, 4, 200.0 106.0 2.5 1.3 2.5 1.3 150 ± 90  79 ± 48 10Y1NH2PMC LPB Bip 3 12.5 5.9 2.5-5   1.2-2.4 2.5 1.2 LFB Bip 6, 8 ≧12.5 ≧5.83.0 1.4 2.5 1.2 LFB Bip 3 PMC ≦25 ≦10.5 2.5 1.1 2.5-5   1.1-2.1 LFB Bip8 PMC 15.0 6.3 2.5 1.1 2.5 1.1 LFB Nal 6, 8 10.0 4.8 20.0 9.6 5.02.4 >1000 >479 LFB Tbt6 12.5 5.6 2.5 1.1   1-2.5 0.4-1.1 2.51.1 >1000 >448 LFB Tbt6 PMC 37.5 15.0 2.5-5   1.0-2.0 5.0 2.0 410 ± 70164 ± 28 LFB Tbt8 12.5 5.6 2.5-5   1.1-2.2 0.5-2.5 0.2-1.1 2.51.1 >1000 >448 LFB Tbt8 PMC 25-50 10-23 2.5-7.5 1.0-3.0 5.0 2.0 290 ± 70116 ± 28 LFB Tbt6, 8   25-37.5 12-18 2.5 1.2 5.0 2.3 2.5 1.2 230 ± 30107 ± 14 LFB Tbt3 25.0 11.1 2.5 1.1 2.5 1.1 500 ± 40 223 ± 18

EXAMPLE 13

[0331] Table 14 below gives MIC values for a variety of peptidesaccording to the invention which incorporate a proportion of D aminoacids. MIC μg/ml MIC μg/ml MIC μg/ml Peptide Sequence* E. coli S. aureusMRSA LFM 17-27 TYR-ARG-ALA- 10 2.5 A7R2, 8, 9W3, 4, 10Y TRP-ARG-TRP- 1NH2 ALA-TRP-ARG- TRP-ARG-CONH2 tyr-ARG-ala-TRP- 7.5 7.5 2.5arg-TRP-ala-TRP- arg-TRP-arg- CONH2 tyr-arg-ala-TRP- 7.5 5 2.5ARG-TRP-ala- TRP-ARG-TRP- ARG-CONH2 LFM 18-24 ARG-TRP-TRP- 10 1 R1, 7W2,3, 6Y5 NH2 ARG-TYR-TRP- ARG-CONH2 arg-TRP-trp-ARG- 7.5 5 2.5tyr-TRP-arg- CONH2 arg-trp-TRP-ARG- 7.5 5 2.5 TYR-trp-arg- CONH2

EXAMPLE 14

[0332] Cytotoxicity of the Peptides of the Invention

[0333] The cytotoxic effect of the peptides on different murine andhuman tumor cells were measured using the MTT method (Mosmann et al., J.Immunol. (1986) 136, 2348-2357). MTT is a soluble tetrazolium saltyielding a yellow solution when prepared in media or salt solutionslacking phenol red. Dissolved MTT is converted to an insoluble purpleformazan by cleavage of the tetrazolium ring by dehydrogenase enzymes.This water insoluble formazan can be solubilized using isopropanol orother solvents and the dissolved material is measuredspectrophotometrically. The absorbance was measured as a function ofconcentration of converted dye.

[0334] The conversion of the soluble dye to the insoluble purpleformazan is utilized in assays for measurement of cell proliferation.Active mitochondrial dehydrogenases of living cells cause this change,while dead cells do not.

[0335] We used this assay to measure the degree of cell death caused bypeptides.

[0336] Cells:

[0337] Cells were maintained in RPMI-1641 medium containing 10% FBS, 1%L-glutamine and 0.1% penicillin and streptomycin. Cells to be used inthe assay were grown to confluency, trypsinated and split to single cellsuspension, counted and centrifuged at 1500 rpm for 10 min. The cellpellet was resuspended to a concentration of 4×10⁵ cells/ml in RPMI-1640without FBS and L-glutamine (assay-medium). 100 ml of cell suspensionwas transferred to each well on a 96-well microtiter plate. The cellswere stimulated by adding 100 ml of various concentrations of peptidesdiluted with assay medium to each well. The final concentrations ofpeptide were for example: 5, 10, 20, 40, 60, 80, 100, and 200 mg/ml.Because there is a twofold dilution upon adding the peptide solution tothe wells containing the cell suspension, the peptide solution had to bemade twofold concentrated. As a negative control only medium was addedto the cells, and as a positive control (100% killing) 1% triton X-100was added. Following an incubation period of 4 h., 20 ml MTT dissolvedin PBS at a concentration of 5 mg/ml was added to each well, and theplate was incubated further for 2 h. 130 ml of the supernatant were thenremoved and 100 ml acid alcohol (0.04-0.1 N HCl in isopropanol) added toeach well to dissolve the dark blue crystals. The plate was placed on ashaker for 1 h and read spectrophotometrically at 590 nm in amicrotiterplate reader using the Softmaxâ program.

[0338] Hemolytic Assay

[0339] The hemolytic activities of the peptides were determined usingfresh human red blood cells. 8 ml blood was taken from a healthy person.4 ml blood was transferred to a polycarbonate tube containing heparin toa final concentration of 10 U/ml, and the remaining 4 ml blood wastransferred to a glass tube containing EDTA with final concentration of15% EDTA. The erythrocytes were isolated from heparin-treated blood bycentrifugation in 1500 rpm for 10 min and washed three times withphosphate-buffered saline (PBS) to remove plasma and buffy coat. Thecell pellet was resuspended in PBS to make the final volume of 4 ml. Thepeptide was diluted to a concentration of 2 mg/ml and 0.1 mg/ml. Thepeptide was further diluted to the concentrations as stated in Table 15.For each tube PBS was added first, then RBCs and peptide solutions. Thehematocrit in the blood treated with EDTA was determined after 30 minwith Sysmex K-1000, and the resuspended RBCs were diluted into 10%hematocrit. RBCs in PBS (1%) with and without peptides (Table 15) wereincubated in a shaker at 370 for 1 hour and then centrifuged at 4000 rpmfor 5 min. The supernatant were carefully transferred to newpolycarbonate tubes and the absorbance of the supernatant was measuredat 540 nm. Baseline hemolysis was hemoglobin released in the presence ofPBS, and 100% hemolysis was hemoglobin released in the presence of 0.1%Triton X-100. TABLE 15 Final peptide Peptide or Total concentrationTriton X-100 Red blood PBS Volume Tube No. (μg/ml) (μl) cells (μl) (μl)(μl) 1 Neg. control 70 630 700 (2 mg/ml peptide) 2 Pos. control 7 70 623700 (2 mg/ml peptide) 3 1000 250 50 200 500 (2 mg/ml peptide) 4 500 12550 325 500 (2 mg/ml peptide) 5 100 35 70 595 700 (2 mg/ml peptide) 6 5017.5 70 612.5 700 (2 mg/ml peptide) 7 10 70 70 560 700 (0.1 mg/mlpeptide) 8 1 7 70 623 700 (0.1 mg/ml peptide)

EXAMPLE 15

[0340] Solid Phase Peptide Synthesis

[0341] Initially the lactoferricin B used was a gift from Wayne Bellamy(NutritiOnal Science Laboratory, Morinaga Milk industry Co. Ltd, Japan).All the other peptides were synthesized on a 9050 Millipore AutomaticPeptide Synthesizer. Generally, in solid phase synthesis, peptide chainsare assembled from the carboxy teminus to the amino acid terminus. Thefirst (C-terminal) amino acid was covalently attached to an insolublesupport (the resin) by a linker (4-hydroxymethyl-phenoxyacetic acid).The remaining amino acids were added, one by one, until the peptidesequence was completed.

[0342] Using the Fmoc method, the a-amino end of the amino acid wastemporary protected by the base labile 9-fluorenylmethoxycarbonyl (Fmoc)group. Not only the α-amino group of the amino acid was protected. Someof the amino acids have reactive side chains which are necessary toprotect during the synthesis to prevent side reactions. These protectinggroups, except for cysteine, were acid labile and cleaved upon treatmentwith TEA (trifluoroacetic acid) and scavengers (see below).

[0343] Prior to the synthesis, to the solid support PEG-PS (PolyEthylene Glycol-Poly Styrene resin) was added a small volume of DMF(dimethylformamide) and left to swell for 30 minutes. Packed into acolumn, the Fmoc group was removed by treatment with a 20% piperidinesolution in DMF. The incomming protected amino acid was now able to bindto the free amino end of the resin-linked amino acid with its carboxyend. However, coupling or acylation does not occur spontanously, thecarboxylate must be activated., This was achieved by the use ofpreactivated amino acids, such as pentafluorophenyl (Pfp) esters, oramino acids with free carboxylates able to react with the couplingreagent HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluoro-phosphate) in situ. Using Pfp esters, 1.3 eq. of HOBt(1-hydroxy-benzotriazole) was added to catalyse the reaction, while whenusing the coupling reagent HATU, the reaction was base catalysed with2.4 eq. of DIPEA (diisopropylethylamine). A four-fold excess ofactivated amino acids was generally employed. The amino acids weredissolved in the activator solution in sufficient quantity, ascalculated by the Express-Peptide program.

[0344] Amino acids were then delivered to the support-bound aminoacid/peptide with fully deprotected α-amine group, and recycled throughthe loop mixed to achieve peptide bond formation. The capacity of theresins used scaled from 0.15 to 0.23 mmol/g, meaning available bindingsites for the incomming amino acids, wherefrom the amount of activatoreqvivalents was calculated. The standard coupling cycle for amino acidswas 30 minutes, with the exception of arginine, isoleucine, threonine,tyrosine, valine, and the amino acids coupled thereafter, requiring 60minutes. Extended coupling times for these amino acids were chosenbecause of their large side chains which are known to cause stericalhindrance during the coupling reaction. Once coupling was complete, theexcess amino acid solution and reaction by-products were removed bywashing with DMF. The next cycle begun with deblocking of the α-aminogroup of the N-teminal amino acid. The process of α-amino groupdeblocking followed by coupling was repeated for as many cycles asnecessary to assemble the desired peptide.

[0345] After the synthesis was complete, the column material wastransferred to a funnel and washed with methanol (3×) anddichloromethane (2×). The cleavage of the acid labile side chainprotecting groups and cleavage of the peptides from the solid supportwas achieved using a mixture of 2% anisol, 2% ethandithiol, 2% water and2% phenol in TFA, and with cleavage times of no more than four hours.The solid support was then removed by filtration, the filtrateconcentrated under a high vacuum and the peptide precipitated in diethylether. The ether solution containing TFA was removed using a pasteurpipette, and the peptide was washed several times with diethyl ether anddried under a high vacuum.

[0346] Amino Acid Derivatives:

[0347] Fmoc-L-Ala-OPfp

[0348] Fmoc-L-Arg(Pmc)-OPfp

[0349] Fmoc-L-Cys(Acm)-OPfp

[0350] Fmoc-L-Gln-OPfp

[0351] Fmoc-L-Glu(OtBu)-OPfp

[0352] Fmoc-L-Gly-OPfp

[0353] Fmoc-L-Ile-OPfp

[0354] Fmoc-L-Leu-OPfp

[0355] Fmoc-L-Lys(tBoc)-OPfp

[0356] Fmoc-L-Met-OPfp

[0357] Fmoc-L-Phe-OPfp

[0358] Fmoc-L-Ser(tBu)-OPfp

[0359] Fmoc-L-Trp-OPfp

[0360] Fmoc-L-Tyr(tBu)-OPfp

[0361] Fmoc-L-Val-OPfp

[0362] Amino Acid Derivatives:

[0363] Amino acid derivatives were purchased from either Bachem,MilliGen/Biosearch (Division of Millipore) or PerSeptive Biosystems.Phenol was purchased from Fluka, and anisole was purchased from Sigma.DMF, PIP, DIPEA TFA and PEG-PS resin were all purchased from PerSeptiveBiosystems.

EXAMPLE 16

[0364] Table 16 below shows the anti-tumour activity and toxic data fora LFB 14-31 derivative incorporating either of two non-genetic bulky andlipohilic amino acids in place of a Trp or incorporating a group (Pmc)which increases the bulk and lipophilicity of one of the naturallyoccuring Trp or Phe residues. TABLE 16 RBC EC₅₀ Meth A EC₅₀ VariablePeptide (μM) (μM) LFB1431A2, 6, 10, 17 >440 165 LFB1431A2, 6, 10, 17Bip4336 23.4 LFB1431A2, 6, 10, 17Pmc 165 12.8 LFB1431A2, 6, 10, 17Tbt9 25.69.5

[0365] The presence of either of the three non-genetically modificationson a LFB 14-31 derivative significantly increased its anti-tumoractivity. The Tbt modified peptide however possesed the highesthemolytic activity among the three modified analogs tested.

EXAMPLE 17

[0366] Table 17 below shows the anti-bacterial and anti-tumouralactivity and toxicity of further peptides according to the invention. Inparticular, the substitutions show how replacement of non-essentialtryptophan residues may result in peptides with advantageously lowtoxicity (activity against red blood cells and normal fibroblasts).TABLE 17 Mic S. Meth A IC₅₀ Mic E-coli Aureus RBC EC₅₀ FibroblastSubstituion Peptide (μM) (4 h) (μM) (μM) (μM) IC₅₀ (μM) LFB14-31A_(2, 6, 10, 17)F₇K₁₆L₁₄R₄ 6.6 2/4 2 110 17 Alanine W3 → A3 LFB14-31A_(2, 3, 6, 10, 17)F₇K₁₆L₁₄R₄ 24.1 15 10 >463 190 W9 → A9 LFB14-31A_(2, 6, 9, 10, 17)F₇K₁₆L₁₄R₄ 16.2 10 5 382 46.3 W11 → A11 LFB14-31A_(2, 6, 10, 11, 17)F₇K₁₆L₁₄R₄ 11.1 10 >2.5 278 46.3 W9, 11 → A9,11 LFB 14-31A_(2, 6, 9, 10, 11, 17)F₇K₁₆L₁₄R₄ 110.1 30 30 >489 >489Lysine W3 → K3 LFB 14-31A_(2, 6, 10, 17)F₇K_(3, 16)L₁₄R₄ 230 >451 230 W9→ K9 LFB 14-31A_(2, 6, 10, 17)F₇K_(9, 16)L₁₄R₄ 13.5 30 10 >451 58.7 W11→ K11 LFB 14-31A_(2, 6, 10, 17)F₇K_(11, 16)L₁₄R₄ 7.9 5 <2.5 >451 30.7W9, 11 → K9, 11 LFB14-31A_(2, 6, 10, 17)F₇K_(9, 11, 16)L₁₄R₄ >300 >463 >463 Isoleucine W3 →I3 LFB 14-31A_(2, 6, 10, 17)F₇I₃K₁₆L₁₄R₄ 9 2/4 2/4 323 20 W9 → I9 LFB14-31A_(2, 6, 10, 17)F₇I₉K₁₆L₁₄R₄ 12 5 <1 26 W11 → I11 LFB14-31A_(2, 6, 10, 17)F₇I₁₁K₁₆L₁₄R₄ 6 2/5 <1 15 W9, 11 → I9, 11 LFB14-31A_(2, 6, 10, 17)F₇I_(9, 11)K₁₆L₁₄R₄ 22 26 W3, 9 → I3, 9 LFB14-31A_(2, 6, 10, 17)F₇I_(3, 9)K₁₆L₁₄R₄ 36 5 5 >470 108 W3, 11 → I3, 11LFB 14-31A_(2, 6, 10, 17)F₇I_(3, 11)K₁₆L₁₄R₄ 16 2.5 5 413 45 W3, 9, 11 →I3, 9, 11 LFB 14-31A_(2, 6, 10, 17)F₇I_(3, 9, 11)K₁₆L₁₄R₄ 47 2.5 10 >487280 F7 → A7 LFB 14-31A_(2, 6, 10, 17)F₇K₁₆L₁₄R₄ 34.6 15 10 >455 288.9

1. A modified lactoferrin peptide which is cytotoxic, 7 to 25 aminoacids in length, with three or more cationic residues and which has oneor more extra bulky and lipophilic amino acids as compared to the nativelactoferrin sequence, as well as esters, amides, salts and cyclicderivatives thereof, wherein the extra bulky and lipophilic amino acidcomprises a bulky and lipophilic R group having 7 or more non-hydrogenatoms but excluding the peptide LFB (17-41) wherein the cysteineresidues are pyridylethylated.
 2. A peptide as claimed in claim 1wherein the extra bulky and lipophilic amino acid is selected from thegroup comprising tyrosine, tryptophan and phenylalanine.
 3. A peptide asclaimed in claim 1 wherein the extra bulky and lipophilic amino acid isa non-genetic amino acid.
 4. A peptide as claimed in claim 3 wherein thenon-genetic amino acid is selected from biphenylalanine,tri-tert-butyltryptophan, 2-naphtylalanine, ortho-nitrophenylsulfinyl,ortho-nitrophenylsulfonyl and adamantylalanine.
 5. A peptide as claimedin claim 3 wherein the non-genetic amino acid is a standard geneticamino acid whose R group has been modified.
 6. A peptide as claimed inclaim 5 wherein the R group is modified by a protecting group.
 7. Apeptide as claimed in cliam 6 wherein the protecting group is selectedfrom the group consisting of Pmc, Pbf, tert-buyl and carboxybenzyl.
 8. Amodified lactoferrin peptide which is cytotoxic, 7 to 25 amino acids inlength, with three or more cationic residues and which has one or moreextra bulky and lipophilic amino acids as compared to the nativelactoferrin sequence, as well as esters, amides, salts and cyclicderivatives thereof, wherein the extra bulky and lipophilic amino acidcomprises a bulky and lipophilic N-terminal group which is a cyclicgroup comprising at least 5 non-hydrogen atoms.
 9. A peptide as claimedin claim 8 wherein the N-terminal group comprises 2 or more fused rings.10. A modified lactoferrin peptide which is cytotoxic, 7 to 25 aminoacids in length, with three or more cationic residues and which has oneor more extra bulky and lipophilic amino acids as compared to the nativelactoferrin sequence, as well as esters, amides, salts and cyclicderivatives thereof, wherein the extra bulky and lipophilic amino acidcomprises a bulky and lipophilic C-terminal group which comprises atleast 4 non-hydrogen atoms.
 11. A pharmaceutical composition comprisinga peptide as claimed in any one of claims 1 to 10, together with aphysiologically acceptable diluent, carrier or excipient.
 12. A peptideas claimed in any one of claims 1 to 10 for use as a medicament.
 13. Amethod of treating bacterial infections in a patient comprising theadministration to said patient of one or more of the peptides claimed inany one of claims 1 to
 10. 14. A method of treating tumours in a patientcomprising the administration to said pateient of one or more of thepeptides claimed in any one of claims 1 to
 10. 15. A method ofinhibiting the growth of bacteria comprising contacting the bacteriawith an inhibiting effective amount of a peptide as claimed in any oneof claims 1 to
 10. 16. A method of enhancing the cytotoxicity orselectivity of a 7 to 25 mer lactoferrin originating peptide with threeor more cationic residues by incorporating therein an extra bulky andlipophilic amino acid as defined in any one of claims 1 to
 10. 17. Apeptide comprising the enantio or retro-enantio fort of LFB or fragmentsthereof.
 18. Use of LFB and fragments thereof in the manufacture of amedicament for the treatment of solid tumours.